A block copolymer and a method for preparing and using the same
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ZHEJIANG UNIV OF TECH
- Filing Date
- 2023-10-25
- Publication Date
- 2026-06-05
Smart Images
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Abstract
Description
Technical Field
[0001] This invention relates to a block copolymer, its preparation method, and its application. Background Technology
[0002] Malignant tumors are a major threat to human life and health, and their incidence and mortality rates are increasing year by year. Globally, in 2022, there were 19.3 million new cases and 10 million deaths from malignant tumors worldwide. Therefore, developing effective treatments for malignant tumors is of great social significance. Currently, the main treatment methods commonly used in clinical practice include surgical resection, chemotherapy, radiotherapy, and immunotherapy. Among these, immunotherapy is a revolutionary cancer treatment method following traditional surgery, chemotherapy, and radiotherapy. It shifts the focus from directly destroying cancer cells to recognizing and attacking cancer cells by activating the host's anti-tumor immune response.
[0003] Currently, several chemotherapy IDO-1 immunotherapy nanomedicines have been designed and some progress has been made. However, these combined therapeutic nanomedicines typically utilize encapsulation or chemical covalent linkage to carry chemotherapy drugs and IDO-1 inhibitors, which easily leads to problems such as low drug loading, premature drug leakage, and complex chemical synthesis, seriously hindering the clinical translation of these drugs. Using supramolecular strategies, the above challenges faced by combined therapeutic nanomedicines can be effectively overcome.
[0004] Therefore, we can utilize supramolecular strategies to construct supramolecular nanomedicines that can not only efficiently carry chemotherapy drugs and IDO-1 inhibitors, but also achieve selective drug release from the tumor microenvironment, thereby enhancing the efficiency of IDO-1 immunotherapy for cancer chemotherapy. Summary of the Invention
[0005] The purpose of this invention is to provide a block copolymer, its preparation method and application, to solve the defects of existing chemotherapy systems in cancer treatment, such as high toxicity and poor immunogenicity.
[0006] The technical solution adopted in this invention is:
[0007] In a first aspect, the present invention provides a block copolymer of Formula 6, denoted as mPEG-PLG-COOH-CD:
[0008] 6, n is 45, m is 4, p is 4.
[0009] In a second aspect, the present invention provides a method for preparing the block copolymer shown in Formula 6, the method comprising the following steps:
[0010]
[0011] (1) Preparation of compound 3: Compound 1 and compound 2 were dissolved in anhydrous dimethylformamide (DMF), and then nitrogen gas was passed through and stirred at room temperature for 24-72 h. The reaction solution was post-treated to obtain compound 3.
[0012]
[0013] (2) Preparation of compound 4: Compound 3 prepared by step (1) was dissolved in dichloroacetic acid, and a 33% hydrobromic acid-acetic acid solution was added. The mixture was stirred at 30°C for 1-10 h. The reaction mixture was added to diethyl ether to precipitate the precipitate. The precipitate was filtered, redissolved in DMF, and dialyzed in deionized water (preferably MWCO 3500 Da) for 26-72 h. The retentate was freeze-dried at 5°C to obtain compound 4.
[0014]
[0015] (3) Preparation of compound 6 (mPEG-PLG-COOH-CD): Compound 4 prepared by step (2) was dissolved in dimethylformamide, and then N-hydroxysuccinimide (NHS), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) were added. The reaction was carried out at room temperature for 12-36 h. After the reaction was completed, the reaction mixture was added dropwise to diethyl ether for precipitation. After filtration, the precipitate was dried under vacuum at 25 °C to obtain the carboxyl-activated polymer. All the carboxyl-activated polymer and β-CD-NH2 were dissolved in DMF, and triethylamine was added. After the reaction was carried out at room temperature for 24 h, the reaction mixture was added dropwise to acetone for precipitation. After filtration, the precipitate was washed three times with acetone and dried under vacuum at 25 °C to obtain compound 6 (mPEG-PLG-COOH-CD).
[0016] Further, in step (1), the molar ratio of compound 1 to compound 2 is 1:5-50 (preferably 1:10); the volume of anhydrous dimethylformamide is 30-100 mL / g (preferably 50 mL / g) based on the mass of compound 1.
[0017] Further, in step (1), the post-treatment method of the reaction solution is as follows: the reaction solution is added to diethyl ether for precipitation, filtered, and the resulting filter cake is vacuum dried at room temperature to obtain compound 3; the volume of the diethyl ether is 10-100 mL / g based on the mass of compound 1, preferably 20 mL / g.
[0018] Further, in step (2), the volume of dichloroacetic acid used is 10-100 mL / g (preferably 10 mL / g) based on the mass of compound 3; the volume of the hydrobromic acid-acetic acid solution used is 1-10 mL / g (preferably 3 mL / g) based on the mass of compound 3; the volume of dimethylformamide used is 1-10 mL / g (preferably 2 mL / g) based on the mass of compound 3; and the volume of diethyl ether used is 10-500 mL / g (preferably 20 mL / g) based on the mass of compound 3.
[0019] Further, in step (3), the volume of dimethylformamide used to dissolve compound 4 is 50-200 mL / mmol (preferably 100 mL / mmol) based on the amount of compound 4; the molar ratio of compound 4 to N-hydroxysuccinimide is 1:1-5, preferably 1:4; the molar ratio of compound 4 to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:5-15, preferably 1:10; the molar ratio of compound 4 to 4-dimethylaminopyridine is 1:1-3, preferably 1:2.3; the molar ratio of compound 4 to β-CD-NH2 is 1:1-5, preferably 1:4; and the volume of triethylamine used is 0.1-1 mL / mmol, preferably 0.2 mL / mmol, based on the amount of compound 4. The volume of dimethylformamide used to dissolve β-CD-NH2 is 200-600 mL / mmol, preferably 500 mL / mmol, based on the molar amount of compound 4. The volume of diethyl ether used is 10-100 mL / g, preferably 20 mL / g, based on the mass of compound 4; the volume of acetone used for precipitation or washing is 10-100 mL / g, preferably 20 mL / g, based on the mass of compound 4.
[0020] Thirdly, the present invention provides the application of the block copolymer in the preparation of antitumor nanomedicines.
[0021] Furthermore, the antitumor nanomedicine uses the block copolymer as a carrier to simultaneously carry Pt-type chemotherapeutic drugs and IDO-1 inhibitors to form supramolecular nanomedicines. Under the action of weak acid within tumor cells, the nanomedicine swells, exposing the drugs, while a high concentration of Cl... - It disrupts the non-covalent bonds within the nanomedicine, rapidly releasing Pt-type chemotherapy drugs and IDO-1 inhibitors, while simultaneously activating chemotherapy and immunotherapy, achieving a highly efficient combination of the two therapies.
[0022] Furthermore, the tumor cells are mouse colon cancer cells CT26.
[0023] Furthermore, the supramolecular nanomedicine is prepared according to the following steps: a Pt-based chemotherapy drug solution is added dropwise to a block copolymer solution, followed by the addition of an IDO-1 inhibitor solution. The mixture is stirred at room temperature for 1 hour, then deionized water is added, and the mixture is sonicated for 60 minutes. Finally, the mixture is heated to 50 °C. o Stirred at C for 48 h, then freeze-dried to obtain supramolecular nanomedicine; the Pt-type chemotherapy drug is cisplatin (CDDP); the IDO-1 inhibitor is eicosapentaenoic acid (Epa).
[0024] Furthermore, the solvents of the Pt-type chemotherapy drug solution, block copolymer solution, and IDO-1 inhibitor solution are capable of dissolving each substance and are miscible with water, and are selected from dimethyl sulfoxide, tetrahydrofuran, and dimethylformamide (DMF), respectively, with DMF being more preferred.
[0025] Further, the concentration of the Pt-type chemotherapy drug solution is 5-10 mg / mL, preferably 12 mg / mL; the concentration of the IDO-1 inhibitor solution is 5-15 mg / mL, preferably 14 mg / mL; the concentration of the block copolymer solution is 10-30 mg / mL, preferably 20 mg / mL; and the volume ratio of the block copolymer solution to the Pt-type chemotherapy drug solution and the IDO-1 inhibitor solution is 1:0.1-1:0.1-1, preferably 1:0.25:0.25.
[0026]
[0027] Epa CDDP
[0028] Thirdly, the present invention provides a tumor microenvironment-responsive supramolecular nanomedicine prepared from the block copolymer.
[0029] Compared with existing technologies, the beneficial effects of this invention are mainly reflected in the following: This invention designs and synthesizes a block copolymer mPEG-PLG-COOH-CD, utilizing the host molecule β-CD with a large cavity structure, guest molecules IDO-1 inhibitors (such as Epa), and Pt-type chemotherapeutic drugs (such as cisplatin), to jointly assemble into supramolecular nanomedicines through hydrophilic-reactive interactions, host-guest recognition, and coordination. The supramolecular nanomedicines simultaneously carry Pt-type chemotherapeutic drugs and IDO-1 inhibitors. Under the action of weak acid within tumor cells, the nanomedicines swell, exposing the drugs, while simultaneously releasing high concentrations of Cl... - This process disrupts the non-covalent bonds within the nanomedicine, rapidly releasing chemotherapy drugs and IDO-1 inhibitors, while simultaneously activating chemotherapy and immunotherapy, achieving a highly effective combination of the two therapies. The in-situ activation of chemotherapy not only directly kills cancer cells but also activates the body's anti-tumor immunity. Attached Figure Description
[0030] Figure 1 It is intermediate compound 3 1 H NMR spectrum.
[0031] Figure 2 It is compound 6. 1 H NMR spectrum.
[0032] Figure 3 The graphs show the particle size changes of supramolecular nanomedicines after incubation in PBS for different times (a) and particle size changes in different solvents (b).
[0033] Figure 4 This is a fluorescence image of supramolecular nanomedicine phagocytosis in cells.
[0034] Figure 5 These are cell viability graphs of supramolecular nanomedicines. a represents the CT cell viability graphs after 24 h and 48 h of incubation with mPEG-PLG-CD; b represents the CT cell viability graphs after 24 h of incubation with Epa, CDDP, CDDP@Epa, and PND.
[0035] Figure 6 This is a schematic diagram of the action of supramolecular nanomedicine. Detailed Implementation
[0036] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:
[0037] The operating temperature for this invention is 25-30℃. Mouse colon cancer cells (CT26 cells) were purchased from Hangzhou Hangsi Biotechnology Co., Ltd.
[0038] Example 1: Preparation of intermediate compound 3
[0039]
[0040] Compound 3 (mPEG-PBLG) diblock copolymer was synthesized using a ring-opening method:
[0041] Compound 2 (benzyl glutamate) (0.6 g, 2.3 mmol) and compound 1 (mPEG-NH2) (0.5 g, 0.23 mmol) were added to 30 mL of anhydrous DMF, and stirred at room temperature under nitrogen atmosphere for 72 h. The product was precipitated in 10 mL of diethyl ether, filtered, and the filter cake was dried under vacuum at room temperature to give 1 g of solid product with a yield of 91%, which was compound 3 (mPEG-PBLG). 1 See H NMR spectrum Figure 1 This proves that the compound was successfully prepared.
[0042] Example 2: Preparation of intermediate compound 4
[0043]
[0044] Compound 3 (5.0 g) prepared by the method in Example 1 was dissolved in 50.0 mL of dichloroacetic acid, and 15.0 mL of hydrobromic acid / acetic acid (33 wt%) was added. The mixture was stirred slowly at 30 °C for 1 h. The reaction product was added to 100 mL of diethyl ether to precipitate the product. The product was filtered, and the precipitate was redissolved in 10.0 mL of DMF and dialyzed in deionized water for 72 h (MWCO 3500 Da). The retentate was lyophilized at 5 °C to obtain 5.4 g of white solid, which is compound 4.
[0045] Example 3: Synthesis of Compound 6 (mPEG-PLG-COOH-CD)
[0046]
[0047] Compound 4 (350 mg, 0.1 mmol) prepared by the method in Example 2 was dissolved in 10 mL of DMF, and NHS (136 mg, 0.4 mmol), EDC (200 mg, 1 mmol), and DMAP (10 mg, 0.23 mmol) were added sequentially. The carboxyl activation reaction was carried out at room temperature for 24 h. After the reaction was completed, the reaction mixture was added dropwise to 7 mL of diethyl ether to precipitate the product. The precipitate was filtered, dried under vacuum at 25 °C, and the carboxyl-activated polymer was obtained.
[0048] The fully carboxyl-activated polymer and β-CD-NH2 (470 mg, 0.4 mmol) were dissolved in 50 mL of DMF, and 20 μL of triethylamine was added. The reaction mixture was reacted at room temperature for 24 h. After the reaction was complete, the reaction mixture was added dropwise to 7 mL of acetone to precipitate the polymer. The precipitate was filtered, washed three times with 7 mL of acetone, and dried under vacuum at 25 °C to obtain 0.61 g of the target polymer, namely compound 6 (mPEG-PLG-COOH-CD). 1 See H NMR spectrum Figure 2 As shown, this proves that the compound was successfully prepared.
[0049] Example 4: Synthesis of supramolecular nanomedicines
[0050] (1) 40 mg of mPEG-PLG-COOH-CD prepared in Example 3 was dissolved in 2 mL of dimethylformamide (DMF) and treated under ultrasonic conditions at 400 M Hz for 60 min to obtain mPEG-PLG-COOH-CD solution.
[0051] (2) Dissolve cisplatin (6 mg) in 0.5 mL DMF to obtain a cisplatin solution.
[0052] (3) Dissolve Epa (7 mg) in 0.5 mL DMF to obtain Epa solution.
[0053] (4) Add all the cisplatin solution from step (2) to all the mPEG-PLG-COOH-CD solution from step (1), then slowly add all the Epa solution from step (3) at a rate of 0.1 mL / s. Then slowly add 5 mL of deionized water to the solution at a rate of 0.1 mL / s. Sonicate the mixture at 400 MHz for 60 min, and then at 50 MHz. o Stirred at C for 48 h, then freeze-dried at 25 °C to obtain 6.8 mg of supramolecular nanomedicine with a particle size of 200 nm, denoted as PND.
[0054] Example 5: Size Characterization of Supramolecular Nanomedicines
[0055] 1. Particle size stability of supramolecular nanomedicines
[0056] 1.8 mg of the supramolecular nanomedicine prepared in Example 4 was dispersed in 39 mL of PBS and ultrasonically dispersed at 400 MHz for 15 min. After incubation at 25 °C for 1, 3, 5, 7, and 9 days, samples were taken and the particle size of the supramolecular nanomedicine was measured using zeta potential analysis. The results are shown in […]. Figure 3 As shown in Figure a, after incubation for 1, 3, 5, 7, and 9 days, the particle size remained around 200 nm with little change, demonstrating the long-term stability of supramolecular nanomedicines.
[0057] 2. The effect of solvent on the particle size of supramolecular nanomedicines
[0058] 5 mg of the supramolecular nanomedicine prepared in Example 4 was dispersed in 50 mL of water, PBS, RPMI 1640 medium, and RPMI 1640 medium containing 10% FBS, respectively, and incubated at 25°C for 1 day. Particle size was determined by zeta potential analysis. Results are shown in [Figure missing]. Figure 3 As shown in Figure b, the supramolecular nanomedicine particle size is 200 nm in various solvents, indicating that the particle size of the supramolecular nanomedicine does not vary significantly in different solvents. Nanoparticles of this size can aggregate at the tumor site through the EPR effect (hyperosmolarity retention effect), thereby improving the therapeutic efficiency of nanomedicines.
[0059] Example 6: Cell phagocytosis experiment of supramolecular nanomedicines
[0060] CT26 cells at 1 × 10 4Cells were seeded at a density of 10 μL / well in 96-well plates and allowed to adhere overnight. The culture medium was discarded, and 10 µL of RPMI 1640 medium containing 10 μg / mL rhodamine and 10 μg / mL PND was added to each well. Cells were incubated at 25°C for 2, 4, and 8 h, respectively. Afterward, 10 µL of fresh 4.0% paraformaldehyde was added to each well for fixation for 15 min. Cells were washed twice with PBS, then stained with DAPI (5 μM) and incubated at room temperature in the dark for 15 min. After washing once with PBS, images were captured using a laser confocal microscope (CLSM) to observe the uptake of PND nanoparticles by the cells.
[0061] like Figure 4 As shown, red fluorescence began to enter the cytoplasm at 2 h, and the red fluorescence in the cytoplasm gradually increased over time, proving that PND can gradually accumulate in the cell over time rather than simply diffuse into molecules.
[0062] Example 7: Cytotoxicity Study of Supramolecular Nanomedicines
[0063] PNDs exhibit efficient cellular uptake and pH-responsive drug release; therefore, the MTT assay was used to evaluate their cytotoxicity. The specific procedure is as follows:
[0064] 1. Effects of PND on CT26 cell viability at different culture times
[0065] CT26 cells at 1 × 10 4 Cells were seeded at a density of [insert density here] in 96-well plates and allowed to adhere overnight. The culture medium was discarded, and 50 µL of serum-free RPMI 1640 medium containing 10 μg / mL PND was added to each well. After incubation at 25°C for 24 h, the medium was removed, and the cells were washed once with PBS. The cells were then placed in 100 µL of RPMI 1640 medium containing 0.5 mg / mL MTT reagent and cultured at 25°C for 4 h. The plate was then overturned to remove the liquid, and 150 µL of DMSO was added to each well to dissolve any crystals formed. Finally, the absorbance of each well was measured at 490 nm using a scanning spectrophotometer, and cell viability was calculated. Figure 5 As shown in Figure a, PND did not exhibit any cytotoxicity even after incubation for 24 h or even 48 h, demonstrating its good biocompatibility.
[0066] 2. Effects of different reagents on the viability of CT26 cells
[0067] CT26 cells at 1 × 10 4Cells were seeded at a density of 10 μg / mL in 96-well plates and allowed to adhere overnight. The culture medium was discarded, and 50 µL of serum-free RPMI 1640 medium containing 10 μg / mL PND, Epa, CDDP, or CDDP@Epa was added to each well. After incubation at 25°C for 24 h, the medium was removed, and the cells were washed once with PBS. The cells were then placed in 100 μL of RPMI 1640 medium containing 0.5 mg / mL MTT reagent and cultured at 25°C for 4 h. The plate was then delaminated to remove the liquid, and 150 μL of DMSO was added to each well to dissolve any crystals formed. Finally, the absorbance of each well was measured at 490 nm using a scanning spectrophotometer, and cell viability was calculated.
[0068] like Figure 5 As shown in Figure b, the nanomedicine co-assembled with Epa, CDDP, and mPEG-PLG-CD exhibits a higher cell-killing ability. This demonstrates that PND has the strongest anti-tumor effect.
Claims
1. A block copolymer, with the structure shown in Formula 6, denoted as mPEG-PLG-COOH-CD: 6, n is 45, m is 4, p is 4.
2. A method for preparing the block copolymer according to claim 1, characterized in that, The method is performed according to the following steps: (1) Preparation of compound 3: Compound 1 and compound 2 were dissolved in anhydrous dimethylformamide, and then nitrogen gas was passed through and stirred at room temperature for 24-72 h. The reaction solution was post-treated to obtain compound 3. (2) Preparation of compound 4: Compound 3 prepared by step (1) was dissolved in dichloroacetic acid, and a 33% hydrobromic acid-acetic acid solution was added. The mixture was stirred at 30°C for 1-10 h. The reaction mixture was added to diethyl ether to precipitate the precipitate. The precipitate was filtered and redissolved in anhydrous dimethylformamide. The precipitate was dialyzed in deionized water for 26-72 h. The threshold was freeze-dried at 5°C to obtain compound 4. (3) Preparation of compound 6: Compound 4 prepared by step (2) was dissolved in dimethylformamide, and then N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine were added. The reaction was carried out at room temperature for 12-36 h. After the reaction was completed, the reaction mixture was added dropwise to diethyl ether for precipitation. After filtration, the precipitate was dried under vacuum at 25 °C to obtain the carboxyl-activated polymer. All the carboxyl-activated polymer and β-CD-NH2 were dissolved in anhydrous dimethylformamide, and triethylamine was added. After the reaction was carried out at room temperature for 24 h, the reaction mixture was added dropwise to acetone for precipitation. After filtration, the precipitate was washed three times with acetone and dried under vacuum at 25 °C to obtain compound 6.
3. The preparation method according to claim 2, characterized in that, In step (1), the molar ratio of compound 1 to compound 2 is 1:5-50; the volume of anhydrous dimethylformamide used is 30-100 mL / g based on the mass of compound 1.
4. The preparation method according to claim 2, characterized in that, In step (1), the post-treatment method of the reaction solution is as follows: the reaction solution is added to diethyl ether for precipitation, filtered, and the resulting filter cake is vacuum dried at room temperature to obtain compound 3; the volume of the diethyl ether is 10-100 mL / g based on the mass of compound 1.
5. The preparation method according to claim 2, characterized in that, In step (2), the volume of dichloroacetic acid used is 10-100 mL / g based on the mass of compound 3; the volume of the hydrobromic acid-acetic acid solution used is 1-10 mL / g based on the mass of compound 3; and the volume of dimethylformamide used is 1-10 mL / g based on the mass of compound 3.
6. The preparation method according to claim 2, characterized in that, In step (3), the volume of dimethylformamide used to dissolve compound 4 is 50-200 mL / mmol based on the amount of compound 4; the molar ratio of compound 4 to N-hydroxysuccinimide is 1:1-5; the molar ratio of compound 4 to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:5-15; the molar ratio of compound 4 to 4-dimethylaminopyridine is 1:1-3; the molar ratio of compound 4 to β-CD-NH2 is 1:1-5; the volume of triethylamine used is 0.1-1 mL / mmol based on the amount of compound 4; and the volume of dimethylformamide used to dissolve β-CD-NH2 is 200-600 mL / mmol based on the amount of compound 4.
7. The use of the block copolymer of claim 1 in the preparation of antitumor nanomedicines.
8. The application as described in claim 7, characterized in that, The anti-tumor nanomedicine is a supramolecular nanomedicine made by using the block copolymer as a carrier and simultaneously carrying Pt-type chemotherapy drugs and IDO-1 inhibitors.
9. The application as described in claim 8, characterized in that, The supramolecular nanomedicine was prepared by the following steps: a Pt-type chemotherapy drug solution was added dropwise to a block copolymer solution, followed by the addition of an IDO-1 inhibitor solution. The mixture was stirred at room temperature for 1 hour, then deionized water was added, and the mixture was sonicated for 60 minutes. The mixture was then stirred at 50 °C for 48 hours and freeze-dried to obtain the supramolecular nanomedicine. The Pt-type chemotherapy drug was cisplatin, and the IDO-1 inhibitor was eicosapentaenoic acid (EPA).
10. A tumor microenvironment-responsive supramolecular nanomedicine prepared from the block copolymer of claim 1.