A ROS-responsive polymer material, drug-loaded nanomicelles and a preparation method thereof

Abstract

The application discloses a ROS-responsive polymer material, a drug-loaded nanomicelle and a preparation method thereof, and belongs to the technical field of medical materials. 113 -b-PCL 11 -b-PB 36 A nanomicelle M-Pal loaded with Palbociclib is prepared by using a solvent evaporation method. The drug-loaded nanomicelle can be stored for a long time after being freeze-dried; and after being added into an aqueous phase and being redispersed, the drug-loaded nanomicelle is still a clear and transparent nanomicelle dispersion. The ROS-responsive freeze-dried drug-loaded nanomicelle solves the storage problem of conventional micelle-type drug-loaded nanomaterials for a long time, provides an effective delivery platform for drugs that are difficult to dissolve in water, has a ROS-responsive drug release characteristic, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical materials technology, specifically relating to a ROS-responsive polymer material, drug-loaded nanomicelles, and their preparation method. Background Technology

[0002] Biocompatible polymers are widely used in the pharmaceutical field. When used for drug delivery, these polymers can improve drug pharmacokinetics, reduce toxic side effects, and significantly enhance therapeutic efficacy. Amphiphilic block polymers can self-assemble into nanomicelles in aqueous media, serving as delivery carriers for water-insoluble drugs. With the development of various novel amphiphilic block polymers, nanomicelle-based drug delivery systems, as a highly promising nanomedicine delivery technology, have been successfully applied in multiple fields.

[0003] Nanometric micelles are thermodynamically stable colloidal aggregates formed by the self-assembly of amphiphilic materials in aqueous media, exhibiting an ordered arrangement. The formation mechanism of nanomicelles is as follows: when the concentration of the amphiphilic material in the aqueous medium exceeds a critical value, hydrophobic groups aggregate due to hydrophobic interactions to form the core of the nanomicelle, while hydrophilic groups maintain their interaction with water molecules, forming the outer shell of the nanomicelle and ensuring its stable dispersion in the aqueous medium.

[0004] Nanomicelles, as drug delivery materials, possess advantages such as small particle size, structural stability, strong drug solubilization ability, and low toxicity, and have been widely used for various drug loading applications. For example, Genexol nanomicelles loaded with paclitaxel were developed by the South Korean biopharmaceutical company Samyang Biopharm. ® PM. The polymer material used in this product is methoxylated polyethylene glycol- b - Poly(D,L-lactide) (MPEG- b -PDLLA). Genexol ® PM is already available in South Korea for the treatment of metastatic breast cancer, non-small cell lung cancer, and ovarian cancer. In addition, the South Korean biopharmaceutical company Samyang Biopharm is developing methoxylated polyethylene glycol-b-poly(D,L-lactide) (MPEG-) b -PDLLA) has developed nanomicelles loaded with docetaxel for polymer materials. ® M is currently undergoing clinical trials for recurrent or metastatic squamous cell carcinoma of the head and neck. Nanoxel ®Clinical trials of cyclosporine A combined with oxaliplatin for the treatment of metastatic esophageal squamous cell carcinoma are also underway. Indian pharmaceutical company Sun Pharma has developed nano-Cequa loaded with cyclosporine A. Cequa promotes tear secretion and has been approved by the US FDA for the treatment of dry eye. Domestic research on drug-loaded nanomicelles has also been reported. Paclitaxel-loaded nanomicelles developed by Shanghai Yizhong Pharmaceutical Co., Ltd. have been approved by the China National Medical Products Administration for use in combination with platinum-based drugs to treat patients with epidermal growth factor receptor gene mutation-negative, anaplastic lymphoma kinase-negative, unresectable locally advanced or metastatic non-small cell lung cancer.

[0005] Although there is considerable research on drug-loaded nanomicelles, the number of nanomicelle products that have ultimately achieved commercialization and clinical application remains limited. The reasons for this include the complexity of the preparation process of amphiphilic polymer materials, the difficulty in quality control, and the challenges in storing the resulting drug-loaded nanomicelles. Among these, long-term storage of drug-loaded nanomicelles is one of the major obstacles to their successful commercialization. Currently, most drug-loaded nanomicelles are stored in aqueous dispersions, which are difficult to store long-term. Lyophilization of ordinary drug-loaded nanomicelles destroys their structure, preventing them from being uniformly redispersed in an aqueous medium. Therefore, developing a class of drug-loaded nanomicelles that can be stored long-term after lyophilization and can be redispersed in an aqueous medium for the delivery of drugs that are poorly soluble in water is of great significance. Summary of the Invention

[0006] The present invention aims to provide a ROS-responsive polymer material, drug-loaded nanomicelles, and a method for preparing the same. These drug-loaded nanomicelles exhibit high drug loading capacity for poorly water-soluble drugs and possess reactive oxygen species-responsive drug release characteristics, making them suitable for the delivery of antitumor drugs. Furthermore, these drug-loaded nanomicelles can be stored long-term after lyophilization and can be redispersed in an aqueous medium, thus solving the long-term storage problem of drug-loaded nanomicelles and demonstrating promising application prospects.

[0007] To achieve the above objectives, the technical method adopted in this application is as follows:

[0008] This 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, poly(ε-caprolactone) (PCL) with different degrees of polymerization m is first block-modified. m ; and then block-modify ROS-responsive poly[4-(acryloyloxymethylene)phenylboronic acid pinacol ester] with different degrees of polymerization n, abbreviated as PB nThe final product is called MPEG. 113 - b -PCL m - b -PB n The structural formula is shown in the following general formula I:

[0009]

[0010] Where m and n are selected from 5 to 100 respectively.

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

[0012]

[0013] Synthesis of 4-(acryloyloxymethylene)-Pinacolyl phenylboronic acid ester. 4-(hydroxymethyl)-phenylboronic acid 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, under nitrogen protection, 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, followed by extraction of the reaction mixture with a saturated aqueous solution of sodium chloride (CAS No. 7647-14-5). After separating the dichloromethane solution, the mixture was concentrated by vacuum distillation. The crude product was purified by silica gel rapid column chromatography to give a white solid, 4-(acryloyloxymethylene)-phenylboronic acid ester.

[0014] MPEG 113 - b -PCL m Synthesis of a similar material. Monomethoxy polyethylene glycol (CAS No. 9004-74-4) with a degree of polymerization of 113 and different equivalents of ε-caprolactone (CAS No. 502-44-3) were dissolved in toluene (CAS No. 108-88-3), followed by the addition of stannous isooctanoate (CAS No. 301-10-0). The reaction mixture was stirred at 105ºC 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. This will be achieved using MPEG. 113 - b -PCL m 2-Bromoisobutyryl bromide (CAS No. 20769-85-1) was dissolved in dichloromethane (CAS No. 75-09-2), and triethylamine (CAS No. 121-44-8) was added. The mixture was stirred at room temperature for 12 hours. The reaction mixture was then added to 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 similar materials. MPEG 113 - b -PCL m -BIBB and 4-(acryloyloxymethylene)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 protection. The reaction mixture was stirred at 60ºC for 24 hours, purified by rapid column chromatography using 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 is as follows. This invention attempts different feeding ratios. Taking different polymer products as examples, the degree of polymerization of each block is as follows: the degree of polymerization of monomethoxy polyethylene glycol MPEG is 113, the degree of polymerization of poly(ε-caprolactone) PCL is 11, and the degree of polymerization of poly[4-(acryloyloxymethylene)phenylboronic acid pinacol ester]PB is 18, 30, 36, and 40.

[0018]

[0019] This invention also discloses a ROS-responsive, lyophilizable drug-loaded nanomicelle, utilizing the aforementioned ROS-responsive polymer material MPEG. 113 -b -PCL m - b -PB n Empty nanomicelles were prepared via nanoprecipitation. Drug-loaded nanomicelles were then prepared via solvent evaporation using a drug loading agent dissolved in an organic solvent and the empty nanomicelles. Finally, the drug-loaded nanomicelles were dehydrated using freeze-drying to obtain ROS-responsive freeze-dryable drug-loaded nanomicelles.

[0020] From MPEG 113 - b -PCL m - b -PB n The best-performing MPEG ratio was selected from the currently screened materials. 113 - b -PCL 11 - b -PB 36 Taking the CDK4 / 6 inhibitor Palbociclib (CAS No. 571190-30-2) as an example, the preparation of ROS-responsive lyophilizable drug-loaded nanomicelles includes the following steps:

[0021] Preparation of empty nanomicelles: MPEG 113 - b -PCL 11 - b -PB 36 Dissolved in acetone (CAS No. 67-64-1), twice the volume of ultrapure water was added under vigorous vortex conditions. After removing the acetone by vacuum distillation, empty nanomicelles were obtained.

[0022] Preparation of drug-loaded nanomicelles: Palbociclib was dissolved in chloroform (CAS No. 67-66-3) and added dropwise to the empty nanomicelles prepared in the above step under vigorous stirring (stirring speed 1000 r / min, stirring time 4 h). Stirring was continued for 2 hours. After the chloroform had evaporated completely, the nanomicelles were dehydrated by freeze drying to obtain ROS-responsive freeze-dryable drug-loaded nanomicelles loaded with Palbociclib, hereinafter referred to as M-Pal.

[0023] The beneficial effects of the above-mentioned technical solution of the present invention are as follows:

[0024] This drug-loaded nanomicelle exhibits high drug loading capacity for poorly water-soluble drugs and reactive oxygen species-responsive drug release characteristics, making it suitable for the delivery of antitumor drugs. Furthermore, the drug-loaded nanomicelles can be stored long-term after lyophilization and can be redispersed in an aqueous medium, thus solving the long-term storage problem of drug-loaded nanomicelles and demonstrating promising application prospects. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 Pinarol ester of 4-(acryloyloxymethylene)phenylboronic acid 1 H-NMR results.

[0027] Figure 2 Pinarol ester of 4-(acryloyloxymethylene)phenylboronic acid 13 C-NMR results.

[0028] Figure 3 The ESI mass spectrometry results are for pinacol ester of 4-(acryloyloxymethylene)phenylboronic acid.

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

[0030] Figure 5 For compound MPEG 113 - b -PCL 11 -BIBB 1 H-NMR results.

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

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

[0033] Figure 8 For compound MPEG 113 - b -PCL 11 - b -PB 36 of1 H-NMR results.

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

[0035] Figure 10 To prepare MPEG loaded with the CDK4 / 6 inhibitor Palbociclib 113 - b -PCL 11 - b -PB 36 The image shows the effect of redispersing drug-loaded nanomicelles M-Pal after lyophilization and storage for 1 day or 30 days in ultrapure water.

[0036] Figure 11 The diagram illustrates the drug loading and encapsulation efficiency of lyophilized M-Pal.

[0037] Figure 12 This is a graph showing the ROS-responsive release characteristics of M-Pal after short-term and long-term storage following freeze-drying.

[0038] Figure 13 The diagram illustrates the particle size and surface potential of M-Pal after one day of freeze-drying (left) and after one month of freeze-drying, when redispersed in ultrapure water (right).

[0039] Figure 14 A diagram illustrating the effects of Palbociclib and M-Pal on the viability of pancreatic cancer cells. Detailed Implementation

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort 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 products or can be prepared by known and established methods.

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

[0043]

[0044] 10 g (0.043 mol) of 4-(hydroxymethyl)phenylboronic acid pinacol ester and 5.2 g (0.051 mol) of triethylamine were dissolved in 10 mL of dichloromethane. After cooling in an ice-water bath under nitrogen protection, acryloyl chloride (4.6 g, 0.051 mol) was added dropwise with stirring. After removing the ice bath, the mixture was stirred at room temperature for 12 hours, followed by extraction of the reaction mixture with a saturated aqueous solution of sodium chloride. The dichloromethane solution was separated and concentrated by vacuum distillation. The crude product was purified by silica gel column chromatography to give a white solid, 4-(acryloyloxymethylene)phenylboronic acid pinacol ester. Figure 1 and Figure 2 These are pinacol esters of 4-(acryloyloxymethylene)phenylboronic acid. 1 H-NMR and 13 C-NMR results. Peaks and their corresponding proton assignments in the spectra are indicated by lowercase letters. The integral values ​​of the methyl proton resonance peak (1.34 ppm) in phenylboronic acid and the proton resonance peaks in the acryloyloxy chain (6.45, 6.17, and 5.85 ppm) were calculated. Figure 3 The ESI mass spectrometry results for pinacol 4-(acryloyloxymethylene)phenylboronic acid pinacol ester are shown. The molecular weight of pinacol 4-(acryloyloxymethylene)phenylboronic acid pinacol ester is 288.153. After binding with Na⁺, it forms an adduct ion [M+Na]⁺ at m / z 311.143. The peak at m / z 311.142 in the mass spectrum corresponds to [M+Na]. + . 1 H NMR (600MHz, CDCl3) δ 7.81 (d, J = 7.5 Hz, 2H), 7.37 (d, J = 7.6 Hz, 2H), 6.45 (dd, J =17.4, 1.3 Hz, 1H), 6.17 (dd, J = 17.3, 10.4 Hz, 1H), 5.85 (dd, J = 10.4, 1.3 Hz,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 Spectrum: [M+Na] calculated for C 16 H 21BO4: 311.143; found 311.142.

[0045] Example 2: Compound MPEG 113 - b -PCL 11 Synthesis

[0046]

[0047] Monomethoxy polyethylene glycol (1 g, molecular weight 5000, degree of polymerization 113, 0.0002 mol) and ε-caprolactone (0.25 g, 0.0022 mol) were dissolved in 1 mL toluene, and then stannous isooctanoate (20 mg) was added. The reaction mixture was stirred at 105ºC for 18 hours. The reaction mixture was then added to petroleum ether, and a white solid MPEG precipitated. 113 - b -PCL 11 . Figure 4 For compound MPEG 113 - b -PCL 11 of 1 H-NMR results. Peaks and their corresponding proton assignments in the spectra are indicated with lowercase letters. The average degree of polymerization of the polycaprolactone (PCL) block is 11, which was calculated by comparing the integral values ​​of the methylene proton resonance peak (3.65 ppm, peak b) in the monomethoxy polyethylene glycol (MPEG) backbone with the methylene proton resonance peaks (peak c: 2.31 ppm, peaks d and f: 1.65 ppm, peak e: 1.39 ppm) in the PCL backbone. 1 H NMR (600 MHz, CDCl3) δ 4.23 (m, 2H), 4.08 – 4.04 (m, 22H), 3.76 (m, 2H), 3.65 (s, 454H), 3.38 (s, 3H), 2.31 (t, J = 7.4 Hz, 22H), 1.68 – 1.62 (m, 44H), 1.39 (m, 22H).

[0048] Example 3: Compound MPEG 113 - b -PCL 11 -BIBB Synthesis

[0049]

[0050] MPEG 113 - b -PCL 115 g (0.00082 mol) of methyl methacrylate (MCMA) and 2-bromoisobutyryl bromide (0.14 g, 0.00061 mol) were dissolved in 5 mL of dichloromethane, and triethylamine (0.06 g, 0.00059 mol) was added. The mixture was 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 For compound MPEG 113 - b -PCL 11 -BIBB 1 ¹H-NMR results. Peaks and their corresponding proton assignments in the spectra are indicated with lowercase letters. The successful coupling of 2-bromoisobutyryl bromide was determined by comparing the terminal methyl proton resonance peak of monomethoxy polyethylene glycol (peak a: 3.38 ppm) with the terminal methyl proton resonance peak of 2-bromoisobutyl (peak h: 1.93 ppm). 1 H-NMR (600 MHz, CDCl3) δ 4.23 (m,2H), 4.17 (m, 1H), 4.07 (m, 21H), 3.76 (m, 2H), 3.67 – 3.62 (m, 453H), 3.38(d, J = 2.9 Hz, 3H), 2.31 (m, 22H), 1.93 (s, 6H), 1.65 (q, J = 6.8 Hz, 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 11BIBB (1 g, 0.00019 mol) and compound 4-(acryloyloxymethylene)phenylboronic acid pinacol ester (1.4 g, 0.0048 mol) were dissolved in 0.56 mL of anisole. Under nitrogen protection, cuprous bromide (0.056 g, 0.00038 mol) and tris[2-(dimethylamino)ethyl]amine (0.045 g, 0.00038 mol) were added. The reaction mixture was stirred at 60ºC for 24 hours, purified by rapid column chromatography using neutral alumina, concentrated, and precipitated in petroleum ether to give a pale gray solid MPEG. 113 - b -PCL 11 - b -PB 18 . Figure 6 For compound MPEG 113 - b -PCL 11 - b -PB 18 of 1 ¹H-NMR results. Peaks and their corresponding proton assignments in the spectra are indicated with lowercase letters. The average degree of polymerization of poly[4-(acryloyloxymethylene)phenylboronic acid pinacol ester] (PB) is 18, which was calculated by comparing the integral values ​​of the terminal methyl proton resonance peak of monomethoxy polyethylene glycol (MPEG) (peak a: 3.38 ppm), the methylene proton resonance peak in the polycaprolactone (PCL) backbone (peak b: 2.35-2.25 ppm), and the benzene ring proton resonance peak of poly[4-(acryloyloxymethylene)phenylboronic acid pinacol ester] (PB) (peak c: 7.73 ppm). 1 H-NMR (600 MHz, CDCl3) δ 7.73 (s, 36H), 7.21 (s, 36H), 4.89 (s, 36H), 4.23 (t, J = 4.9 Hz, 2H), 4.06 (t, J = 6.7 Hz, 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 -PCL 11 - b -PB30 Synthesis

[0055]

[0056] Compound MPEG 113 - b PC 11 BIBB (1 g, 0.00019 mol) and compound 4-(acryloyloxymethylene)phenylboronic acid pinacol ester (2.8 g, 0.0096 mol) were dissolved in 0.88 mL of anisole. Under nitrogen protection, cuprous bromide (0.056 g, 0.00038 mol) and tris[2-(dimethylamino)ethyl]amine (0.045 g, 0.00038 mol) were added. The reaction mixture was stirred at 60ºC for 24 hours, purified by rapid column chromatography using neutral alumina, concentrated, and precipitated in petroleum ether to give a pale gray solid MPEG. 113 - b -PCL 11 - b -PB 30 . Figure 7 For compound MPEG 113 - b -PCL 11 - b -PB 30 of 1 H-NMR results. Peaks and their corresponding proton assignments in the spectra are indicated with lowercase letters. The average degree of polymerization of poly[4-(acryloyloxymethylene)phenylboronic acid pinacol ester] (PB) is 30, which was calculated by comparing the integral values ​​of the terminal methyl proton resonance peak of monomethoxy polyethylene glycol (MPEG) (peak a: 3.38 ppm), the methylene proton resonance peak in the polycaprolactone (PCL) backbone (peak b: 2.35-2.25 ppm), and the benzene ring proton resonance peak of poly[4-(acryloyloxymethylene)phenylboronic acid pinacol ester] (PB) (peak c: 7.73 ppm). 1 H-NMR (600 MHz, CDCl3) δ 7.73 (s, 61 H), 7.21 (s, 61H), 4.89 (s, 60 H), 4.23 (t, J = 4.9 Hz, 2H), 4.06 (t, J= 6.7 Hz, 18H), 3.82 –3.49 (m, 397 H), 3.38 (s, 3H), 2.35 – 2.25 (m, 22H), 2.08 – 1.80 (m, 31 H),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-(acryloyloxymethylene)phenylboronic acid pinacol ester (2.8 g, 0.0096 mol) were added without 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 60ºC for 24 hours, purified by rapid column chromatography using neutral alumina, concentrated, and precipitated in petroleum ether to give a pale gray solid MPEG. 113 - b -PCL 11 - b -PB 36 . Figure 8 For compound MPEG 113 - b -PCL 11 - b -PB 36 of 1 H-NMR results. Peaks and their corresponding proton assignments in the spectra are indicated with lowercase letters. The average degree of polymerization of poly[4-(acryloyloxymethylene)phenylboronic acid pinacol ester] (PB) is 36, which was calculated by comparing the integral values ​​of the terminal methyl proton resonance peak of monomethoxy polyethylene glycol (MPEG) (peak a: 3.38 ppm), the methylene proton resonance peak in the polycaprolactone (PCL) backbone (peak b: 2.35-2.25 ppm), and the benzene ring proton resonance peak of poly[4-(acryloyloxymethylene)phenylboronic acid pinacol ester] (PB) (peak c: 7.73 ppm). 1H-NMR (600 MHz, CDCl3) δ 7.73 (s, 72 H), 7.21 (s, 79H), 4.89 (s, 72 H), 4.23 (t, J = 4.9 Hz, 3 H), 4.06 (t, J = 6.7 Hz, 18H), 3.82 – 3.49 (m,393 H), 3.38 (s, 3H), 2.35 – 2.25 (m, 22 H), 2.08 – 1.80 (m, 75 H), 1.75 –1.52 (m, 23 H), 1.49 – 1.20 (m, 421 H).

[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-(acryloyloxymethylene)phenylboronic acid pinacol ester (4.2 g, 0.0144 mol) were dissolved in 1.21 mL of anisole. 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 60ºC for 24 hours, purified by rapid column chromatography using neutral alumina, concentrated, and precipitated in petroleum ether to give a dark gray solid MPEG. 113 - b -PCL 11 - b -PB 40 . Figure 9 For compound MPEG 113 - b -PCL 11 - b -PB 40 of 1¹H-NMR results. Peaks and their corresponding proton assignments in the spectra are indicated with lowercase letters. The average degree of polymerization of poly[4-(acryloyloxymethylene)phenylboronic acid pinacol ester] (PB) is 40, which was calculated by comparing the integral values ​​of the terminal methyl proton resonance peak of monomethoxy polyethylene glycol (MPEG) (peak a: 3.38 ppm), the methylene proton resonance peak in the polycaprolactone (PCL) backbone (peak b: 2.35-2.25 ppm), and the benzene ring proton resonance peak of poly[4-(acryloyloxymethylene)phenylboronic acid pinacol ester] (PB) (peak c: 7.73 ppm). 1 H-NMR (600 MHz, CDCl3) δ 7.73 (s, 80H), 7.21 (s, 80 H), 4.89 (s, 80 H), 4.23 (t, J = 4.9 Hz, 2 H), 4.06 (t, J = 6.7 Hz, 17 H), 3.82 – 3.49 (m, 389 H), 3.38 (s, 3H), 2.35 – 2.25 (m, 22 H), 2.08 – 1.80 (m, 31 H), 1.75 – 1.52 (m, 60 H), 1.49 – 1.20 (m, 601 H).

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

[0064] From MPEG 113 - b -PCL m - b -PB n The best-performing MPEG ratio was selected from the currently screened materials. 113 - b -PCL 11 - b -PB 36 Taking the CDK4 / 6 inhibitor Palbociclib as an example, the preparation includes the following steps:

[0065] (1) Preparation of empty nanomicelles. Weigh 100 mg of MPEG 113 - b -PCL 11 - b -PB 36 Dissolved in 2 ml of acetone, 4 ml of water was added under vigorous vortex. After removing the acetone by vacuum distillation, empty nanomicelles were obtained.

[0066] (2) Preparation of drug-loaded nanomicelles. 10 mg of Palbociclib was dissolved in 3 ml of chloroform and added dropwise to the empty nanomicelles described in (1) under vigorous stirring. After stirring for 2 hours, the chloroform was completely evaporated and then dehydrated by freeze-drying to obtain ROS-responsive freeze-dryable drug-loaded nanomicelles loaded with Palbociclib, hereinafter referred to as M-Pal.

[0067] Results analysis: such as Figure 10 After being freeze-dried and stored for 1 day or 30 days, the micelles M-Pal, when redispersed in ultrapure water, produced a clear and transparent dispersion with no precipitate.

[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. Mobile phase: 30% acetonitrile and 70% aqueous solution (containing 0.1% trifluoroacetic acid); Detection wavelength: 254 nm; Column temperature: 30 ºC. Sample preparation: First, 0, 25, 50, 100, and 200 μg / mL standards of Palbociclib were prepared for standard curve determination. Then, 1 mg of lyophilized micelles was weighed, dissolved in 1 mL of dimethyl sulfoxide, and the drug content was measured.

[0070] Results analysis: such as Figure 11 The micelle M-Pal containing palbociclib had a drug loading of 7.98 ± 0.79% (by weight) and an encapsulation efficiency of 79.8 ± 7.85%, demonstrating high drug loading and encapsulation efficiency.

[0071] Example 10: Reactive Oxygen Response Drug Load Release Test of M-Pal

[0072] Figure 12 The cumulative drug release curves of M-Pal after lyophilization for one day (left) and after lyophilization for one month and redispersed in ultrapure water (right) are shown in PBS containing hydrogen peroxide (H2O2) and PBS without H2O2, measuring the micellar reactive oxygen species responsive loading release capacity.

[0073] Take M-Pal after lyophilization and storage for 1 day and 1 month, and disperse it in ultrapure water. Add phosphate buffer (PBS) or PBS containing 100 μM hydrogen peroxide (H2O2) and conduct drug release experiments in a 37 ºC water bath.

[0074] Results analysis: Both short-term and long-term storage of freeze-dried M-Pal showed good ROS-responsive release characteristics. There was no significant difference in drug release characteristics among M-Pal stored for different durations after freeze-drying.

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

[0076] 50 mg of M-Pal, which had been lyophilized and stored for 1 day and 1 month respectively, were dispersed in 2 ml of ultrapure water and vortexed to prepare samples. After appropriate dilution, the particle size and potential of the micelles were measured using dynamic light scattering (ZetaSizer ZS90, Malvern Instrument, UK).

[0077] Results analysis: such as Figure 13 Both freeze-dried M-Pal and those stored for short and long periods exhibit small particle size and surface potential. After freeze-drying, M-Pal showed no significant difference in particle size and surface potential after different storage times.

[0078] Example 12: Assay of pancreatic cancer cell viability

[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, MIAPaCa-2, and mouse primary pancreatic cancer cells KPC-A548 at 37 ºC for 72 hours. Cell viability was measured using the CCK-8 assay. The half-inhibitory concentration (IC50) was used. 50 The inhibitory effects of Palbociclib and M-Pal on pancreatic cancer cell viability were characterized.

[0080] Results analysis: such as Figure 14 Palbociclib and M-Pal have similar inhibitory effects on pancreatic cancer cell viability.

[0081] Terminology definitions and explanations:

[0082] Unless otherwise stated, the definitions of groups and terms recorded in this application specification and claims, including their definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, and definitions of specific compounds in the examples, can be arbitrarily combined and combined with each other. Such combinations and combinations of group definitions and compound structures shall fall within the scope of this application specification.

[0083] Unless otherwise stated, the numerical ranges described in this specification and claims correspond to at least each of the specific integer values ​​described herein. For example, the numerical range "1-10" corresponds to each of the integer values ​​in the numerical range "1-10", namely 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. It should be understood that when describing substituents herein, "more than" as one, two, or more should refer to integers ≥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 within the protection scope 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 monomethoxy polyethylene glycol MPEG with a degree of polymerization of 113 113 First, poly(ε-caprolactone) with a degree of block polymerization m is synthesized, followed by poly[4-(acryloyloxymethylene)phenylboronic acid pinacol ester] with a degree of block polymerization n, and finally prepared, with the structural formula as shown in general formula I: ， Where m is 11 and n is 36.

2. A method for preparing the ROS-responsive polymer material as described in claim 1, characterized in that, The preparation steps of the compound of general formula I are as follows: (1) Synthesis of 4-(acryloyloxymethylene)pinacol ester: 4-(hydroxymethyl)pinacol ester and triethylamine were dissolved in dichloromethane, an organic solvent. After cooling in an ice-water bath, nitrogen protection was applied, and 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. After separating the dichloromethane solution, the mixture was concentrated by vacuum distillation. The crude product was purified by silica gel column chromatography to obtain a white solid 4-(acryloyloxymethylene)pinacol ester. (2) MPEG 113 - b -PCL m Synthesis: Monomethoxy polyethylene glycol with a degree of polymerization of 113 and ε-caprolactone were dissolved in toluene, and then stannous isooctanoate was added. The reaction system was stirred at 105ºC for 18 hours. Afterwards, the reaction mixture was added to petroleum ether to precipitate and obtain MPEG. 113 - b -PCL m ; (3) MPEG 113 - b -PCL m -BIBB synthesis: combining MPEG 113 - b -PCL m 2-Bromoisobutyryl bromide was dissolved in dichloromethane, and triethylamine was added. The mixture was stirred at room temperature for 12 hours. The reaction mixture was then added to petroleum ether to precipitate MPEG. 113 - b -PCL m -BIBB; (4) MPEG 113 - b -PCL m - b -PB n Synthesis: MPEG 113 - b -PCL m -BIBB and 4-(acryloyloxymethylene)phenylboronic acid pinacol ester were dissolved in anisole. Cuprous bromide and tris[2-(dimethylamino)ethyl]amine were added under nitrogen protection. The reaction mixture was stirred at 60ºC for 24 hours, purified by neutral alumina 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 nanomicelles, characterized in that, Firstly, the ROS-responsive polymer material MPEG described in claim 1 is utilized. 113 - b -PCL m - b -PB n Empty nanomicelles were prepared by nanoprecipitation, and drug-loaded nanomicelles were prepared by solvent evaporation using drug loading dissolved in organic solvent and empty nanomicelles. Finally, the drug-loaded nanomicelles were dehydrated by freeze-drying to obtain ROS-responsive freeze-dryable drug-loaded nanomicelles.

4. A method for preparing ROS-responsive lyophilizable drug-loaded nanomicelles as described in claim 3, characterized in that, Includes the following steps: Preparation of empty nanomicelles: MPEG 113 - b -PCL 11 - b -PB 36 Dissolved in acetone, with twice the volume of ultrapure water added, and the acetone removed by vacuum distillation, empty nanomicelles were obtained. Preparation of drug-loaded nanomicelles: Palbociclib was dissolved in chloroform and added dropwise to empty nanomicelles under stirring. After stirring for 2 hours, the chloroform was completely evaporated and then dehydrated by freeze-drying to obtain ROS-responsive freeze-dryable drug-loaded nanomicelles loaded with the CDK4 / 6 inhibitor Palbociclib.

5. The method for preparing ROS-responsive lyophilizable drug-loaded nanomicelles according to claim 4, characterized in that, The method involves dissolving Palbociclib in chloroform and adding it dropwise into empty nanomicelles under stirring at a speed of 1000 r / min for 4 h.

Images