A hyperbranched polyglycerol drug-loaded bottlebrush polymer and a preparation method thereof

Abstract

The application provides a drug-loaded bottle-brush polymer of hyperbranched polyglycerol and a preparation method thereof, and relates to the technical field of bottle-brush polymers.The application utilizes Grubbs-III catalyst to perform water-phase ring-opening metathesis polymerization on a hyperbranched macromonomer to obtain a bottle-brush polymer; an excessive amount of the bottle-brush polymer is reacted to convert the hydroxyl groups in the bottle-brush polymer into carboxyl groups; and then, a grafting reaction is performed with paclitaxel to obtain the drug-loaded bottle-brush polymer of hyperbranched polyglycerol.The bottle-brush hPG synthesized by the application is a safe and efficient nanodrug carrier, and has great potential to replace expensive albumin.In addition, the bottle-brush hPG has a large number of -OH groups and excellent hydrophilicity, so that it becomes a multifunctional polymer platform coupled with other drugs or targeting molecules.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bottle brush polymer, in particular to a hyperbranched polyglycerol drug-loaded bottle brush polymer and a preparation method thereof. BACKGROUND

[0002] Paclitaxel (PTX) is a broad-spectrum anticancer drug used to treat various cancers such as breast cancer and pancreatic cancer (PDAC). PTX itself is hydrophobic and not suitable for direct intravenous injection. In clinical practice, human serum albumin is often used as a nanocarrier for PTX (nab-PTX), but the formulation is too expensive, especially for patients in developing countries. There is still a great demand for a hydrophilic, low-cost carrier that can be produced by synthetic chemistry.

[0003] Ring-opening metathesis polymerization (ROMP) is an important tool for synthesizing bottle brush polymers through the "graft-through" method. The polymerization is active and can occur under high viscosity conditions, with monomer conversion higher than 90%. Bottle brush polymers with good biocompatibility and high drug loading efficiency have always been needed in the biomedical field.

[0004] Hyperbranched polyglycerol (HPG) is a polymer of glycerol with high hydrophilicity, abundant -OH groups that can be modified, and good biocompatibility. It even has a stronger ability to prevent protein corona formation than PEG. Therefore, synthesizing bottle brush hPG and using it as a drug carrier has great application prospects. SUMMARY

[0005] To overcome the above problems, the present application provides a hyperbranched polyglycerol drug-loaded bottle brush polymer and a preparation method thereof. The present application starts from O-(2-aminoethyl) polyethylene glycol (H2N-PEG-OH), the -NH2 group at the end is first protected by triphenylmethyl (Trt), and then the -OH group is initiated by CsOH to undergo anionic ring-opening polymerization of PG; followed by ROMP, a bottle brush hPG with a degree of polymerization of 200 is obtained, wherein the abundant -OH groups in hPG are the reaction sites for succinic anhydride and PTX modification. The PTX loaded by the bottle brush polymer of the present application has significantly enhanced antitumor activity compared to PTX loaded by hPG.

[0006] The preparation method of the hyperbranched polyglycerol drug-loaded bottle brush polymer of the present application comprises the following steps:

[0007] (1) Water-phase ring-opening metathesis polymerization of hyperbranched macromonomer using Grubbs-III catalyst to obtain a bottle brush polymer;

[0008] (2) the hydroxyl groups in the bottlebrush polymer are converted into carboxyl groups by reacting with an excess of the bottlebrush polymer, and then grafting with paclitaxel to obtain a ROMP drug-loaded bottlebrush polymer of hyperbranched polyglycerol;

[0009] The structural formula of the hyperbranched macromonomer is as follows:

[0010]

[0011] In the formula, n = 6-227, and g, m, and k are each independently 10-60.

[0012] Preferably, the synthesis method of the hyperbranched macromonomer MM-4 according to the present application is as follows:

[0013] S1, amino protection of H2N-EG 22 -OH to obtain Trt-NH-EG 22 -OH;

[0014] S2, anionic ring-opening polymerization of Trt-NH-EG 22 -OH with glycidol to obtain Trt-NH-EG 22 -hPG 55 ;

[0015] S3, deprotection of the Trt-NH-EG 22 -hPG 55 using trifluoroacetic acid to obtain NH2-EG 22 -hPG 55 ;

[0016] S4, reaction of the NH2-EG 22 -hPG 55 with norbornene-NHS ester to obtain the hyperbranched macromonomer MM-4; the structural formula of the norbornene-NHS ester is as follows:

[0017]

[0018] Preferably, the synthesis step of step S2 is as follows:

[0019] 0.176 g of cesium hydroxide monohydrate is dissolved in 30 mL of benzene, and 1.0 g of Trt-NH-EG 22-OH. After stirring at 90°C for 3h, the solvent was removed by vacuum double-tube. 25 mL of glycidol was added to 5.0 g CaH2 and stirred overnight to remove water, and anhydrous glycidol was obtained by distillation under reduced pressure at 60°C, 15 mmHg. After removing oxygen by freezing, 6.9 mL of anhydrous glycidol was added dropwise to the reaction system in which the solvent was removed. After stirring at 90°C for 24h, methanol and acidic alumina were added, and after dissolution, the mixture was filtered and rotary evaporated to obtain a polymerization mixture. The mixture was separated and purified by flash chromatography column to obtain a solution of compound 1, and the solvent was removed by rotary evaporation to obtain compound 1 Trt-NH-EG 22 -hPG.

[0020] Preferably, the synthesis steps of step S3 are as follows:

[0021] Trt-NH-EG 22 -hPG 0.25g was dissolved in 0.4 mL of dichloromethane and 0.4 mL of trifluoroacetic acid, and stirred in an ice bath for 24h. The solvent and trifluoroacetic acid were removed by vacuum, and the product was frozen and centrifuged in n-hexane for 3 times, and dried in a vacuum drying oven overnight to obtain a brown oily liquid NH2-EG 22 -hPG.

[0022] Preferably, the synthesis steps of step S4 are as follows:

[0023] 0.0252 mmol Norb-GABA-NHS and 0.0203 mmol NH2-EG 22 -hPG was dissolved in 0.4 mL of N,N-dimethylformamide and reacted at room temperature for 48h. The solvent was removed by rotary evaporation, the crude product was dissolved in cold ethanol, and the insoluble material was removed by filtration. The solvent was removed by rotary evaporation, and the precipitate was obtained by low-temperature centrifugation in n-hexane, and then dried in a vacuum drying oven overnight to obtain a brown oily liquid MM-4, whose structural formula is as follows: Norb-EG 22 -hPG

[0024] In one embodiment, the structural formulas of the hyperbranched macromonomers MM-9, MM-11, MM-13, MM-15, MM-17, MM-19, MM-21, MM-23, MM-25, MM-27 described in the present application are as follows:

[0025] MM-9: Norb-EG 6-hPG

[0026] MM-11: Norb-EG 9-hPG

[0027] MM-13: Norb-EG 13 -hPG

[0028] MM-15: Norb-EG 18 -hPG

[0029] MM-17: Norb-EG 45 -hPG

[0030] MM-19: Norb-EG 68 -hPG

[0031] MM-21: Norb-EG 77 -hPG

[0032] MM-23: Norb-EG 113 -hPG

[0033] MM-25: Norb-EG 227 -hPG

[0034] MM-27: Norb-(CH2) 22 -hPG

[0035] Preferably, step (1) of the present application is to remove water from the hyperbranched macromonomer by freezing, to obtain anhydrous hyperbranched macromonomer; the hyperbranched macromonomer is dissolved in dichloromethane, Grubbs-III catalyst is added, and the reaction is stirred at room temperature for 1-2 hours; after the reaction is completed, ethyl vinyl ether is added to terminate the reaction, and then rotary evaporation is performed to remove the solvent, to obtain the polymerization mixture; the polymerization mixture is dissolved in methanol, centrifugal separation is performed to obtain a bottle brush polymer solution, and rotary evaporation is performed to remove the solvent to obtain a brown oily liquid, namely the bottle brush polymer.

[0036] Further preferably, the amount of Grubbs-III catalyst added in step (1) is 0.5%-2% of the mass of the hyperbranched macromonomer.

[0037] Preferably, step (2) of the present application is synthesized as follows:

[0038] The bottle brush polymer is dissolved in N,N-dimethylformamide, succinic anhydride and 4-dimethylaminopyridine are added, and the reaction is stirred at room temperature for 10-30 hours; suction filtration, washing and drying are performed to obtain a precipitate; the precipitate, paclitaxel, N,N'-dicyclohexyl carbodiimide and 4-dimethylaminopyridine are dissolved in N,N-dimethylformamide, and the reaction is performed at room temperature for 20-30 hours; suction filtration, washing and drying are performed to obtain off-white solid, namely the drug-loaded bottle brush polymer of hyperbranched polyglycerol.

[0039] Further preferably, the molar ratio of the bottle brush polymer, succinic anhydride and 4-dimethylaminopyridine in step (2) is 1:5000-30000:0.01.

[0040] Further preferably, the molar ratio of the precipitate, paclitaxel, N,N'-dicyclohexyl carbodiimide, 4-dimethylaminopyridine in step (2) of the present application is 1:300-6000:300-6000:30-600.

[0041] Compared with the prior art, the present application has the following beneficial effects:

[0042] The present application designs a simple method for directly synthesizing hydrophilic bottle brush polymer with hyperbranched polyglycerol (hPG) side chains by ROMP. The quenching effect of -OH groups on Grubbs catalyst is not obvious, and the DP (polymerization degree) of Norb-PEG-hPG macromonomer (MM-4) can reach about 200. Before loading PTX, the bottle brush hPG and its macromonomer show similar negligible cytotoxicity to PEG. When the DP of hPG is 55, the saturation loading rate of hPG macromonomer with succinic anhydride as a connecting arm is 7 PTX per hPG. The obtained BB-5-PTX is completely soluble in water. In terms of net PTX content, the anti-tumor activity of BB-5-PTX is at the same level as that of PTX itself. The endocytosis study shows that the endocytosis amount of the elongated drug-carrying BB-5-PTX is significantly higher than that of its macromonomer MM-4-PTX or ordinary bottle brush BB-5. One bottle brush molecule (DP=47) carries PTX (or Cy3) units into cells at a time, which is much more efficient than 47 individual macromonomer molecules. PI and Annexin V-FITC flow cytometry confirm that the apoptosis mechanism of all PTX conjugated polymers is the same as that of PTX itself; the bottle brush hPG synthesized by the present application is a safe and efficient nanomedicine carrier, which has great potential to replace expensive albumin. In addition, the bottle brush hPG has a large number of -OH groups and excellent hydrophilicity, making it a multifunctional polymer platform for coupling with other drugs or targeting molecules. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 NMR spectrum of BB-5-PTX (8);

[0044] Figure 2 NMR spectrum of PTX;

[0045] Figure 3 Flow cytometry was used to determine the fluorescence intensity of Cyanine3 in PANC-1 cells after treatment with MM-4, BB-5, MM-4-PTX, and BB-5-PTX for 2 hours, 6 hours, and 12 hours. In all data sets, the net PTX concentration was 15 μM. Data are presented as mean ± standard deviation, n=3. **p<0.01, ***p<0.001, ****p<0.0001.

[0046] Figure 4Apoptosis rate of PANC-1 cells after treatment with MM-4, BB-5, PTX, MM-4-PTX, BB-5-PTX (PTX concentration of 15 μΜ and 30 μΜ) for 12 hours.

[0047] Figure 5 Apoptosis rate of PANC-1 cells after treatment with MM-4, BB-5, PTX, MM-4-PTX, BB-5-PTX (PTX concentration of 15 μΜ and 30 μΜ) for 24 hours.

[0048] Figure 6 Apoptosis rate of PANC-1 cells after treatment with MM-4, BB-5, PTX, MM-4-PTX, BB-5-PTX (PTX concentration of 15 μΜ and 30 μΜ) for 24 hours.

[0049] Figure 7 Apoptosis rate of MDA-MB-231 cells after treatment with MM-4, BB-5, PTX, MM-4-PTX, BB-5-PTX (PTX concentration of 15 μΜ and 30 μΜ) for 12 hours.

[0050] Figure 8 Apoptosis rate of MDA-MB-231 cells after treatment with MM-4, BB-5, PTX, MM-4-PTX, BB-5-PTX (PTX concentration of 15 μΜ and 30 μΜ) for 24 hours. All data are expressed as mean ± standard deviation of three independent experiments. * indicates P < 0.05 compared with the control group; # indicates P < 0.001 compared with MM-4 of the same concentration; & indicates P < 0.0001 compared with BB-5 of the same concentration. DETAILED DESCRIPTION

[0051] In order to make the objects, technical solutions and advantages of the present application clearer, the preferred embodiments of the present application will be further described in detail below with reference to the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0052] Example 1

[0053] A drug-loaded hyperbranched polyglycerol bottlebrush polymer is synthesized by the following steps:

[0054] (1) Synthesis of Trt-NH-EG 22 -OH

[0055] 1.8635g N2H-EG 22 -OH was dissolved in 5 mL of dichloromethane, 0.189 g of triethylamine was added to form solution 1. 0.572 g of triphenylmethyl chloride was dissolved in 3 mL of dichloromethane, and the solution was added dropwise to solution 1 under ice bath. After 2.5 h of reaction at room temperature, the solvent was removed by rotary evaporation, and the product was dissolved in toluene and filtered to remove insoluble materials. The crude product was purified by flash chromatography to obtain Trt-NH-EG 22 -OH was dissolved in 5 mL of dichloromethane, 0.189 g of triethylamine was added to form solution 1. 0.572 g of triphenylmethyl chloride was dissolved in 3 mL of dichloromethane, and the solution was added dropwise to solution 1 under ice bath. After 2.5 h of reaction at room temperature, the solvent was removed by rotary evaporation, and the product was dissolved in toluene and filtered to remove insoluble materials. The crude product was purified by flash chromatography to obtain Trt-NH-EG 22 -OH.

[0056] (2) Synthesis of Trt-NH-EG 22 -hPG 55

[0057] 0.176 g of cesium hydroxide monohydrate was dissolved in 30 mL of benzene, and 1.0 g of Trt-NH-EG 22 -OH was dissolved in 5 mL of dichloromethane, 0.189 g of triethylamine was added to form solution 1. 0.572 g of triphenylmethyl chloride was dissolved in 3 mL of dichloromethane, and the solution was added dropwise to solution 1 under ice bath. After 2.5 h of reaction at room temperature, the solvent was removed by rotary evaporation, and the product was dissolved in toluene and filtered to remove insoluble materials. The crude product was purified by flash chromatography to obtain Trt-NH-EG 22 -hPG.

[0058] (3) Synthesis of NH2-EG 22 -hPG 55

[0059] Trt-NH-EG 22 -hPG 0.25 g was dissolved in 0.4 mL of dichloromethane and 0.4 mL of trifluoroacetic acid, and the mixture was stirred under ice bath for 24 h. The solvent and trifluoroacetic acid were removed by vacuum evaporation, and the product was centrifuged in n-hexane for 3 times and dried in a vacuum oven overnight to obtain brown oily liquid NH2-EG 22 -hPG.

[0060] (4) Synthesis of hyperbranched macromonomer MM-4;

[0061] 0.0252 mmol of Norb-GABA-NHS and 0.0203 mmol of NH2-EG 22- hPG was dissolved in 0.4 mL of N,N-dimethylformamide and reacted at room temperature for 48 h. The solvent was removed by rotary evaporation, and the crude product was dissolved in cold ethanol and filtered to remove insoluble materials. The solvent was removed by rotary evaporation, and the precipitate was obtained by centrifugation in n-hexane at low temperature. The precipitate was dried in a vacuum oven overnight to obtain a brown oily liquid, which was the hyperbranched macromonomer MM-4.

[0062] (5) Synthesis of bottlebrush polymer BB-5

[0063] 0.0595 g of bottlebrush polymer was dissolved in 1.0 mL of N,N-dimethylformamide, 0.131 g of succinic anhydride and 0.8 g of 4-dimethylaminopyridine were added, and the mixture was stirred at room temperature for 20 h. The solvent was removed by vacuum suction, a small amount of dichloromethane was added, and the insoluble materials were removed by filtration. The dichloromethane was removed by rotary evaporation, and the precipitate was obtained by centrifugation in diethyl ether at low temperature. The precipitate was dried in a vacuum oven overnight to obtain a brown viscous liquid. 0.0393 g of the viscous liquid, 0.094 g of paclitaxel, 0.0227 g of N,N'-dicyclohexyl carbodiimide, and 0.001 g of 4-dimethylaminopyridine were dissolved in 0.8 mL of N,N-dimethylformamide, and the mixture was reacted at room temperature for 24 h. The product was treated as described above, and a gray-white solid was obtained by drying, which was the ROMP drug-loaded bottlebrush polymer of hyperbranched polyglycerol.

[0064] (6) Synthesis of BB-5-PTX

[0065] 0.0595 g of bottlebrush polymer was dissolved in 1.0 mL of N,N-dimethylformamide, 0.131 g of succinic anhydride and 0.8 g of 4-dimethylaminopyridine were added, and the mixture was stirred at room temperature for 20 h. The solvent was removed by vacuum suction, a small amount of dichloromethane was added, and the insoluble materials were removed by filtration. The dichloromethane was removed by rotary evaporation, and the precipitate was obtained by centrifugation in diethyl ether at low temperature. The precipitate was dried in a vacuum oven overnight to obtain a brown viscous liquid. 0.0393 g of the viscous liquid, 0.094 g of paclitaxel, 0.0227 g of N,N'-dicyclohexyl carbodiimide, and 0.001 g of 4-dimethylaminopyridine were dissolved in 0.8 mL of N,N-dimethylformamide, and the mixture was reacted at room temperature for 24 h. The product was treated as described above, and a gray-white solid was obtained by drying, which was the ROMP drug-loaded bottlebrush polymer of hyperbranched polyglycerol.

[0066] Example 2

[0067] A drug-loaded bottlebrush polymer of hyperbranched polyglycerol was synthesized according to the following steps:

[0068] Steps (1) to (4) were the same as in Example 1,

[0069] Step (5) was the synthesis of bottlebrush polymer BB-6, which was as follows:

[0070] 43.6 mg MM-4 was dissolved in 1 ml benzene, and anhydrous MM-4 was obtained by freeze drying with vacuum double-tube. MM-4 was dissolved in 0.02 mL dichloromethane, and 0.0742 mg Grubbs-III catalyst was added to the solution. After 1 h, the reaction was terminated by adding one drop of ethyl vinyl ether, and then the solvent was removed by rotary evaporation. The polymerization mixture was dissolved in 5 mL methanol, and the bottlebrush polymer solution was obtained by centrifugation with an ultrafiltration centrifuge tube. The solvent was removed by rotary evaporation to obtain a brown oily liquid, which was bottlebrush polymer BB-6.

[0071] Step (6) is the synthesis of BB-6-PTX, which is as follows:

[0072] 0.0232 g BB-6 was dissolved in 1.0 mL N,N-dimethylformamide, and 0.0442 g succinic anhydride and 0.0003 g 4-dimethylaminopyridine were added to the solution. The reaction was stirred at room temperature for 20 h. The solvent was removed by vacuum extraction, and a small amount of dichloromethane was added. The insoluble matter was removed by filtration, and then the dichloromethane was removed by rotary evaporation. The precipitate was obtained by centrifugation in ethyl ether at low temperature. The precipitate was dried in a vacuum drying oven overnight to obtain a brown viscous liquid. 0.0208 g of the viscous liquid, 0.0444 g paclitaxel, 0.0107 g N,N'-dicyclohexyl carbodiimide, and 0.001 g 4-dimethylaminopyridine were dissolved in 0.8 mL N,N-dimethylformamide, and the reaction was carried out at room temperature for 24 h. The product was obtained by the same treatment as before, and the dried product was a gray-white solid, which was BB-6-PTX.

[0073] Example 3

[0074] A drug-loaded bottlebrush polymer of hyperbranched polyglycerol was synthesized according to the following steps:

[0075] Steps (1) to (4) are the same as in Example 1,

[0076] Step (5) is the synthesis of bottlebrush polymer BB-7, which is as follows:

[0077] 50.6 mg MM-4 was dissolved in 1 ml benzene, and anhydrous MM-4 was obtained by freeze drying with vacuum double-tube. MM-4 was dissolved in 0.02 mL dichloromethane, and 0.0425 mg Grubbs-III catalyst was added to the solution. After 1 h, the reaction was terminated by adding one drop of ethyl vinyl ether, and then the solvent was removed by rotary evaporation. The polymerization mixture was dissolved in 5 mL methanol, and the bottlebrush polymer solution was obtained by centrifugation with an ultrafiltration centrifuge tube. The solvent was removed by rotary evaporation to obtain a brown oily liquid, which was bottlebrush polymer BB-7.

[0078] Step (6) is the synthesis of BB-7-PTX, which is as follows:

[0079] 0.0222 g of the bottlebrush polymer was dissolved in 1.0 mL of N,N-dimethylformamide, 0.0487 g of succinic anhydride and 0.0003 g of 4-dimethylaminopyridine were added, and the reaction was stirred at room temperature for 20 h. The solvent was removed by vacuum suction, a small amount of dichloromethane was added, and the insoluble matter was removed by filtration, followed by removal of dichloromethane by rotary evaporation, and the precipitate was obtained by centrifugation at low temperature in diethyl ether. A brown viscous liquid was obtained by drying in a vacuum drying oven overnight. 0.0158 g of the viscous liquid, 0.0378 g of paclitaxel, 0.0091 g of N,N'-dicyclohexyl carbodiimide, and 0.001 g of 4-dimethylaminopyridine were dissolved in 0.8 mL of N,N-dimethylformamide, and the reaction was carried out at room temperature for 24 h. The treatment method was the same as before, and a grayish white solid was obtained by drying, which was BB-7-PTX.

[0080] Example 4

[0081] A drug-loaded bottlebrush polymer of hyperbranched polyglycerol was synthesized according to the following steps:

[0082] (1) Synthesis of Trt-NH-EG6-OH

[0083] 1.0 g of N2H-EG6-OH was dissolved in 5 mL of dichloromethane, and 0.337 g of triethylamine was added to obtain solution 1. 1.022 g of triphenylmethyl chloride was dissolved in 3 mL of dichloromethane, and the solution was added dropwise to solution 1 under ice bath. After reaction at room temperature for 2.5 h, the solvent was removed by rotary evaporation, and the insoluble matter was removed by filtration after dissolving in toluene, and the crude product was obtained by rotary evaporation. The solution of Trt-NH-EG6-OH was obtained by separation and purification by flash chromatography column, and a white solid was obtained by rotary evaporation to obtain Trt-NH-EG6-OH.

[0084] (2) Synthesis of Trt-NH-EG6-hPG 55

[0085] 0.403 g of cesium hydroxide monohydrate was dissolved in 30 mL of benzene, and 1.0 g of Trt-NH-EG6-OH was added. After stirring at 90°C for 3 h, the solvent was removed by vacuum double-tube suction. 25 mL of glycidol was added to 5.0 g of CaH2 and stirred overnight to remove water, and anhydrous glycidol was obtained by distillation under reduced pressure at 60°C and 15 mmHg, and 15.9 mL of anhydrous glycidol was added dropwise to the reaction system after removing oxygen by freezing. After stirring at 90°C for 24 h, methanol and acidic alumina were added, and the polymer mixture was obtained by dissolving and filtering and rotary evaporation. The solution of compound 1 was obtained by separation and purification by flash chromatography column, and the solvent was removed by rotary evaporation to obtain the compound Trt-NH-EG6-hPG.

[0086] (3) Synthesis of NH2-EG6-hPG 55

[0087] Trt-NH-EG6-hPG 1.0 g was dissolved in 0.8 mL dichloromethane and 0.8 mL trifluoroacetic acid, and the reaction was stirred in an ice bath for 24 h. The solvent and trifluoroacetic acid were removed by vacuum, and the product was frozen and centrifuged in n-hexane three times and dried in a vacuum oven overnight to give a brown oily liquid NH2-EG6-hPG.

[0088] (4) Synthesis of hyperbranched macromonomer MM-9;

[0089] 0.209 mmol Norb-GABA-NHS and 0.116 mmol NH2-EG6-hPG were dissolved in 0.8 mL N,N-dimethylformamide and reacted at room temperature for 48 h. The solvent was removed by rotary evaporation, and the crude product was dissolved in cold ethanol and filtered to remove insoluble materials. The solvent was removed by rotary evaporation, and the precipitate was obtained by low-temperature centrifugation in n-hexane and then dried in a vacuum oven overnight to give a brown oily liquid, which was hyperbranched macromonomer MM-9.

[0090] (5) Synthesis of bottlebrush polymer BB-10

[0091] 0.250 g MM-9 was dissolved in 3 mL benzene, and water was removed by vacuum double-tube freezing to give anhydrous MM-9. MM-9 was dissolved in 0.08 mL dichloromethane, and 0.96 mg Grubbs-III catalyst was added and stirred to react. After 1 h, one drop of ethyl vinyl ether was added to terminate the reaction, and then the solvent was removed by rotary evaporation. The polymerization mixture was dissolved in 5 mL methanol, and the bottlebrush polymer solution was separated by centrifugation in an ultrafiltration centrifuge tube, and the solvent was removed by rotary evaporation to give a brown oily liquid, which was bottlebrush polymer BB-10.

[0092] (6) Synthesis of BB-10-PTX

[0093] 0.0538 g bottlebrush polymer was dissolved in 1.0 mL N,N-dimethylformamide, and 0.131 g succinic anhydride and 0.8 mg 4-dimethylaminopyridine were added and stirred to react at room temperature for 20 h. The solvent was removed by vacuum, a small amount of dichloromethane was added, and insoluble materials were removed by filtration, and then dichloromethane was removed by rotary evaporation, and the precipitate was obtained by low-temperature centrifugation in diethyl ether. The precipitate was dried in a vacuum oven overnight to give a brown viscous liquid. 0.100 g of the viscous liquid, 0.0586 g paclitaxel, 0.0142 g N,N'-dicyclohexyl carbodiimide, and 0.001 g 4-dimethylaminopyridine were dissolved in 0.8 mL N,N-dimethylformamide, and the reaction was performed at room temperature for 24 h. The product was treated as described above, and a dried off-white solid was obtained, which was a ROMP drug-loaded bottlebrush polymer of hyperbranched polyglycerol.

[0094] Example 5

[0095] A drug-loaded bottlebrush polymer of hyperbranched polyglycerol was synthesized according to the following steps:

[0096] (1) Synthesis of Trt-NH-EG9-OH

[0097] 1.0 g of N2H-EG9-OH was dissolved in 5 mL of dichloromethane, 0.253 g of triethylamine was added to form solution 1. 0.767 g of triphenylmethyl chloride was dissolved in 3 mL of dichloromethane, and the solution was added dropwise to solution 1 under ice bath. After 2.5 h of reaction at room temperature, the solvent was removed by rotary evaporation, and the product was dissolved in toluene and filtered to remove insoluble matter. The crude product was separated and purified by flash chromatography column to obtain a solution of Trt-NH-EG9-OH, and the solvent was removed by rotary evaporation to obtain white solid, which was Trt-NH-EG9-OH.

[0098] (2) Synthesis of Trt-NH-EG9-hPG 55

[0099] 0.340 g of cesium hydroxide monohydrate was dissolved in 30 mL of benzene, and 1.0 g of Trt-NH-EG9-OH was added. After stirring at 90 °C for 3 h, the solvent was removed by vacuum double-tube pumping. 25 mL of glycidol was added to 5.0 g of CaH2, and the mixture was stirred overnight to remove water. Anhydrous glycidol was obtained by distillation under reduced pressure at 60 °C and 15 mmHg, and after removing oxygen by freezing, 13.4 mL of anhydrous glycidol was added dropwise to the reaction system in which the solvent had been removed. After stirring at 90 °C for 24 h, methanol and acidic aluminum oxide were added, and the mixture was dissolved, filtered, and rotary evaporated to obtain a polymerization mixture. The mixture was separated and purified by flash chromatography column to obtain a solution of compound 1, and the solvent was removed by rotary evaporation to obtain compound Trt-NH-EG9-hPG.

[0100] (3) Synthesis of NH2-EG9-hPG 55

[0101] Trt-NH-EG9-hPG 1.0 g was dissolved in 0.8 mL of dichloromethane and 0.8 mL of trifluoroacetic acid, and the mixture was stirred under ice bath for 24 h. The solvent and trifluoroacetic acid were removed by vacuum pumping, and the product was frozen and centrifuged in n-hexane for 3 times, and then dried in a vacuum drying oven overnight to obtain brown oily liquid NH2-EG9-hPG.

[0102] (4) Synthesis of hyperbranched macromonomer MM-11;

[0103] 0.205 mmol of Norb-GABA-NHS and 0.114 mmol of NH2-EG9-hPG were dissolved in 0.8 mL of N,N-dimethylformamide, and the mixture was reacted at room temperature for 48 h. The solvent was removed by rotary evaporation, and the crude product was dissolved in cold ethanol and filtered to remove insoluble matter. The solvent was removed by rotary evaporation, and the precipitate was obtained by low-temperature centrifugation in n-hexane, and then dried in a vacuum drying oven overnight to obtain brown oily liquid, which was hyperbranched macromonomer MM-11.

[0104] (5) Synthesis of Bottle Brush Polymer BB-12

[0105] 0.250 g of MM-11 was dissolved in 3 mL of benzene and anhydrous MM-11 was obtained by vacuum double-tube freeze-drying. MM-11 was dissolved in 0.08 mL of dichloromethane and 0.94 mg of Grubbs-III catalyst was added and stirred. After 1 h, the reaction was terminated by adding one drop of ethyl vinyl ether and the solvent was removed by rotary evaporation. The polymerization mixture was dissolved in 5 mL of methanol and centrifuged in an ultrafiltration centrifuge tube to obtain a bottle brush polymer solution. The solvent was removed by rotary evaporation to obtain a brown oily liquid, which was bottle brush polymer BB-12.

[0106] (6) Synthesis of BB-12-PTX

[0107] 0.050 g of bottle brush polymer was dissolved in 1.0 mL of N,N-dimethylformamide, 0.119 g of succinic anhydride and 0.7 mg of 4-dimethylaminopyridine were added and stirred at room temperature for 20 h. The solvent was removed by vacuum suction, a small amount of dichloromethane was added, and the insoluble matter was removed by filtration, and then the dichloromethane was removed by rotary evaporation, and the precipitate was obtained by centrifugation in ethyl ether at low temperature. It was dried in a vacuum drying oven overnight to obtain a brown viscous liquid. 0.100 g of the viscous liquid, 0.058 g of paclitaxel, 0.0014 g of N,N'-dicyclohexyl carbodiimide, and 0.001 g of 4-dimethylaminopyridine were dissolved in 0.8 mL of N,N-dimethylformamide and reacted at room temperature for 24 h. The treatment method was the same as before, and a gray-white solid was obtained by drying, which was a ROMP drug-loaded bottle brush polymer of hyperbranched polyglycerol.

[0108] Example 6

[0109] A ROMP drug-loaded bottle brush polymer of hyperbranched polyglycerol was synthesized as follows:

[0110] (1) Synthesis of Trt-NH-EG 13 -OH

[0111] 1.0 g of N2H-EG 13 -OH was dissolved in 5 mL of dichloromethane, and 0.169 g of triethylamine was added to obtain solution 1. 0.511 g of triphenylmethyl chloride was dissolved in 3 mL of dichloromethane, and the solution was added dropwise to solution 1 under ice bath. After reacting at room temperature for 2.5 h, the solvent was removed by rotary evaporation, and then dissolved in toluene to remove the insoluble matter by filtration, and then rotary evaporation was performed to obtain the crude product. The crude product was separated and purified by flash chromatography column to obtain a solution of Trt-NH-EG 13 -OH, and the solvent was removed by rotary evaporation to obtain white solid, which was Trt-NH-EG 13 -OH.

[0112] (2) Synthesis of Trt-NH-EG 13 -hPG55

[0113] 0.259 g of cesium hydroxide monohydrate was dissolved in 30 mL of benzene, and 1.0 g of Trt-NH-EG was added. 13 -OH. After stirring at 90°C for 3 hours, the solvent was removed by vacuum double-row tube extraction. 25 mL of glycidyl ether was added to 5.0 g of CaH2 and stirred overnight to remove water. Anhydrous glycidyl ether was obtained by vacuum distillation at 60°C and 15 mmHg. After deoxygenation by freezing, 10.2 mL of anhydrous glycidyl ether was added dropwise to the solvent-removed reaction system. After stirring at 90°C for 24 hours, methanol and acidic alumina were added. After dissolution, the mixture was filtered and rotary evaporated to obtain the polymerization mixture. The mixture was purified by flash chromatography to obtain a solution of compound 1. The solvent was removed by rotary evaporation to obtain compound Trt-NH-EG. 13 -hPG.

[0114] (3) Synthesis of NH2-EG 13 -hPG 55

[0115] Trt-NH-EG 13 1.0 g of -hPG was dissolved in 0.8 mL of dichloromethane and 0.8 mL of trifluoroacetic acid, and the mixture was stirred in an ice bath for 24 h. The solvent and trifluoroacetic acid were removed under vacuum, and the product was centrifuged three times in hexane and dried overnight in a vacuum drying oven to obtain a brown oily liquid, NH2-EG. 13 -hPG.

[0116] (4) Synthesis of hyperbranched macromonomer MM-13;

[0117] 0.196 mmol Norb-GABA-NHS and 0.109 mmol NH2-EG 13 -hPG was dissolved in 0.8 mL of N,N-dimethylformamide and reacted at room temperature for 48 h. The solvent was removed by rotary evaporation, and the crude product was dissolved in cold ethanol. The insoluble matter was removed by filtration. The solvent was removed by rotary evaporation, and the precipitate was obtained by low-temperature centrifugation in n-hexane. The precipitate was then dried overnight in a vacuum drying oven to obtain a brown oily liquid, which is the hyperbranched macromonomer MM-13.

[0118] (5) Synthetic bottle brush polymer BB-14

[0119] 0.250 g MM-13 was dissolved in 3 mL benzene and anhydrous MM-13 was obtained by vacuum double-tube freeze-drying. MM-13 was dissolved in 0.08 mL dichloromethane and 0.90 mg Grubbs-III catalyst was added to the solution. After 1 h, the reaction was terminated by adding one drop of ethyl vinyl ether and the solvent was removed by rotary evaporation. The polymerization mixture was dissolved in 5 mL methanol and the bottlebrush polymer solution was obtained by centrifugation in an ultrafiltration centrifuge tube. The solvent was removed by rotary evaporation to obtain a brown oily liquid, which was the bottlebrush polymer BB-14.

[0120] (6) Synthesis of BB-14-PTX

[0121] 0.050 g bottlebrush polymer was dissolved in 1.0 mL N,N-dimethylformamide, 0.114 g succinic anhydride and 0.7 mg 4-dimethylaminopyridine were added and the reaction was stirred at room temperature for 20 h. The solvent was removed by vacuum suction, a small amount of dichloromethane was added, and the insoluble material was removed by filtration. The dichloromethane was removed by rotary evaporation, and the precipitate was obtained by centrifugation in ether at low temperature. The precipitate was dried in a vacuum drying oven overnight to obtain a brown viscous liquid. 0.100 g of the viscous liquid, 0.057 g paclitaxel, 0.014 g N,N'-dicyclohexylcarbodiimide, and 0.001 g 4-dimethylaminopyridine were dissolved in 0.8 mL N,N-dimethylformamide, and the reaction was stirred at room temperature for 24 h. The product was obtained by the same method as described above, and the dried product was a gray-white solid, which was the ROMP drug-loaded bottlebrush polymer of hyperbranched polyglycerol.

[0122] Example 7

[0123] A drug-loaded bottlebrush polymer of hyperbranched polyglycerol was synthesized according to the following steps:

[0124] (1) Synthesis of Trt-NH-EG 18 -OH

[0125] 1.0 g N2H-EG 18 -OH was dissolved in 5 mL dichloromethane, and 0.127 g triethylamine was added to obtain solution 1. 0.383 g triphenylmethyl chloride was dissolved in 3 mL dichloromethane, and the solution was added dropwise to solution 1 under ice bath. After the reaction at room temperature for 2.5 h, the solvent was removed by rotary evaporation, and the insoluble material was removed by filtration after dissolution in toluene. The crude product was obtained by rotary evaporation, and the product was purified by flash chromatography column to obtain a solution of Trt-NH-EG 18 -OH, and the white solid obtained by rotary evaporation of the solvent was Trt-NH-EG 18 -OH.

[0126] (2) Synthesis of Trt-NH-EG 18 -hPG 55

[0127] 0.209 g of cesium hydroxide monohydrate was dissolved in 30 mL of benzene, and 1.0 g of Trt-NH-EG was added. 18 OH. After stirring at 90 °C for 3 h, the solvent was removed by vacuum double-tube freezing. 25 mL of glycidol was added to 5.0 g of CaH2, and stirring was continued overnight to remove water. Anhydrous glycidol was obtained by distillation under reduced pressure at 60 °C and 15 mmHg, and oxygen was removed by freezing. Then, 8.3 mL of anhydrous glycidol was added dropwise to the reaction system from which the solvent had been removed. After stirring at 90 °C for 24 h, methanol and acidic alumina were added, and the solution was filtered and rotary evaporated to obtain a polymerization mixture. The mixture was separated and purified by flash chromatography to obtain a solution of compound 1, and the solvent was removed by rotary evaporation to obtain compound Trt-NH-EG 18 -hPG.

[0128] (3) Synthesis of NH2-EG 18 -hPG 55

[0129] Trt-NH-EG 18 -hPG 1.0 g was dissolved in 0.8 mL of dichloromethane and 0.8 mL of trifluoroacetic acid, and stirring was continued at an ice bath for 24 h. The solvent and trifluoroacetic acid were removed by vacuum, and the product was frozen and centrifuged in n-hexane three times, and then dried in a vacuum drying oven overnight to obtain brown oily liquid NH2-EG 18 -hPG.

[0130] (4) Synthesis of hyperbranched macromonomer MM-15;

[0131] 0.188 mmol of Norb-GABA-NHS and 0.104 mmol of NH2-EG 18 -hPG was dissolved in 0.8 mL of N,N-dimethylformamide, and stirring was continued at room temperature for 48 h. The solvent was removed by rotary evaporation, and the crude product was dissolved in cold ethanol, and then filtered to remove insoluble substances. The solvent was removed by rotary evaporation, and the precipitate was obtained by low-temperature centrifugation in n-hexane, and then dried in a vacuum drying oven overnight to obtain brown oily liquid, which was hyperbranched macromonomer MM-15.

[0132] (5) Synthesis of bottlebrush polymer BB-16

[0133] 0.250 g of MM-15 was dissolved in 3 mL of benzene, and anhydrous MM-15 was obtained by vacuum double-tube freezing. MM-15 was dissolved in 0.08 mL of dichloromethane, and 0.87 mg of Grubbs-III catalyst was added and stirred. After 1 h, one drop of ethyl vinyl ether was added to terminate the reaction, and then the solvent was removed by rotary evaporation. The polymerization mixture was dissolved in 5 mL of methanol, and centrifugation was performed using an ultrafiltration centrifuge tube to obtain a bottlebrush polymer solution, and the solvent was removed by rotary evaporation to obtain brown oily liquid, which was bottlebrush polymer BB-16.

[0134] (6) Synthesis of BB-16-PTX

[0135] 0.050 g of bottlebrush polymer was dissolved in 1.0 mL of N,N-dimethylformamide, 0.110 g of succinic anhydride and 0.7 mg of 4-dimethylaminopyridine were added, and the reaction was stirred at room temperature for 20 h. The solvent was removed under vacuum, a small amount of dichloromethane was added, and the insoluble matter was removed by filtration, followed by removal of dichloromethane by rotary evaporation, and the precipitate was obtained by centrifugation at low temperature in diethyl ether. A brown viscous liquid was obtained by drying in a vacuum drying oven overnight. 0.100 g of the viscous liquid, 0.056 g of paclitaxel, 0.014 g of N,N'-dicyclohexyl carbodiimide, and 0.001 g of 4-dimethylaminopyridine were dissolved in 0.8 mL of N,N-dimethylformamide, and the reaction was carried out at room temperature for 24 h. The treatment method was the same as before, and a grayish-white solid was obtained by drying, which was the ROMP drug-loaded bottlebrush polymer of hyperbranched polyglycerol.

[0136] Example 8

[0137] A drug-loaded bottlebrush polymer of hyperbranched polyglycerol was synthesized as follows:

[0138] (1) Synthesis of Trt-NH-EG 45 -OH

[0139] 1.0 g of N2H-EG 45 -OH was dissolved in 5 mL of dichloromethane, 0.051 g of triethylamine was added, and solution 1 was obtained. 0.153 g of triphenylmethyl chloride was dissolved in 3 mL of dichloromethane, and the solution was added dropwise to solution 1 under ice bath. After reaction at room temperature for 2.5 h, the solvent was removed by rotary evaporation, and the insoluble matter was removed by filtration after dissolution in toluene, and the crude product was obtained by rotary evaporation. The solution of Trt-NH-EG 45 -OH was obtained by flash column chromatography, and white solid was obtained by rotary evaporation of the solvent, which was Trt-NH-EG 45 -OH.

[0140] (2) Synthesis of Trt-NH-EG 45 -hPG 55

[0141] 0.097 g of cesium hydroxide monohydrate was dissolved in 30 mL of benzene, and 1.0 g of Trt-NH-EG 45-OH. After stirring at 90 °C for 3 h, the solvent was removed by vacuum double-tube. 25 mL of glycidol was added to 5.0 g CaH2 and stirred overnight to remove water, and anhydrous glycidol was obtained by distillation under reduced pressure at 60 °C, 15 mmHg. After removing oxygen by freezing, 3.8 mL of anhydrous glycidol was added dropwise to the reaction system in which the solvent was removed. After stirring at 90 °C for 24 h, methanol and acidic alumina were added, and after dissolution, the mixture was filtered and rotary evaporated to obtain a polymerization mixture. The solution of compound 1 was separated and purified by flash chromatography column, and the solvent was removed by rotary evaporation to obtain compound Trt-NH-EG 45 -hPG.

[0142] (3) Synthesis of NH2-EG 45 -hPG 55

[0143] Trt-NH-EG 45 -hPG 1.0 g was dissolved in 0.8 mL of dichloromethane and 0.8 mL of trifluoroacetic acid, and stirred in an ice bath for 24 h. The solvent and trifluoroacetic acid were removed by vacuum, and the product was frozen and centrifuged in n-hexane for 3 times, and dried in a vacuum drying oven overnight to obtain a brown oily liquid NH2-EG 45 -hPG.

[0144] (4) Synthesis of hyperbranched macromonomer MM-17;

[0145] 0.150 mmol Norb-GABA-NHS and 0.083 mmol NH2-EG 45 -hPG was dissolved in 0.8 mL of N,N-dimethylformamide and reacted at room temperature for 48 h. The solvent was removed by rotary evaporation, and the crude product was dissolved in cold ethanol, and the insoluble matter was removed by filtration. The solvent was removed by rotary evaporation, and the precipitate was obtained by low-temperature centrifugation in n-hexane, and then dried in a vacuum drying oven overnight to obtain a brown oily liquid, which was hyperbranched macromonomer MM-17.

[0146] (5) Synthesis of bottlebrush polymer BB-18

[0147] 0.250 g of MM-17 was dissolved in 1 mL of benzene, and anhydrous MM-17 was obtained by vacuum double-tube freezing to remove water. MM-17 was dissolved in 0.1 mL of dichloromethane, and 0.70 mg of Grubbs-III catalyst was added and stirred. After 1 h, a drop of ethyl vinyl ether was added to terminate the reaction, and then the solvent was removed by rotary evaporation. The polymerization mixture was dissolved in 5 mL of methanol, and the bottlebrush polymer solution was separated by centrifugation in an ultrafiltration centrifuge tube, and the solvent was removed by rotary evaporation to obtain a brown oily liquid, which was bottlebrush polymer BB-18.

[0148] (6) Synthesis of BB-18-PTX

[0149] 0.050 g bottlebrush polymer was dissolved in 1.0 mL of N,N-dimethylformamide, 0.089 g succinic anhydride and 0.5 mg of 4-dimethylaminopyridine were added, and the reaction was stirred at room temperature for 20 h. The solvent was removed by vacuum evaporation, a small amount of dichloromethane was added, and the insoluble matter was removed by filtration, followed by removal of dichloromethane by rotary evaporation, and the precipitate was obtained by centrifugation at low temperature in diethyl ether. The brown viscous liquid was dried in a vacuum drying oven overnight to obtain a brown viscous liquid. 0.100 g of the viscous liquid, 0.050 g of paclitaxel, 0.012 g of N,N'-dicyclohexyl carbodiimide, and 0.001 g of 4-dimethylaminopyridine were dissolved in 0.8 mL of N,N-dimethylformamide, and the reaction was carried out at room temperature for 24 h. The treatment method was the same as before, and a grayish white solid was obtained by drying, which was the ROMP drug-loaded bottlebrush polymer of hyperbranched polyglycerol.

[0150] Example 9

[0151] A drug-loaded bottlebrush polymer of hyperbranched polyglycerol was synthesized according to the following steps:

[0152] (1) Synthesis of Trt-NH-EG 68 -OH

[0153] 1.0 g of N2H-EG 68 -OH was dissolved in 5 mL of dichloromethane, 0.034 g of triethylamine was added to obtain solution 1. 0.102 g of triphenylmethyl chloride was dissolved in 3 mL of dichloromethane, and the solution was added dropwise to solution 1 under ice bath. After reaction at room temperature for 2.5 h, the solvent was removed by rotary evaporation, and the insoluble matter was removed by filtration after dissolution in toluene, and the crude product was obtained by rotary evaporation. The solution of Trt-NH-EG 68 -OH was obtained by flash column chromatography, and the white solid obtained by rotary evaporation of the solvent was Trt-NH-EG 68 -OH.

[0154] (2) Synthesis of Trt-NH-EG 68 -hPG 55

[0155] 0.067 g of cesium hydroxide monohydrate was dissolved in 30 mL of benzene, and 1.0 g of Trt-NH-EG 68 -OH was added. After stirring at 90°C for 3 h, the solvent was removed by vacuum double-tube evaporation. 25 mL of glycidol was added to 5.0 g of CaH2, and stirring was carried out overnight to remove water. Anhydrous glycidol was obtained by distillation under reduced pressure at 60°C and 15 mmHg, and after oxygen removal by freezing, 2.7 mL of anhydrous glycidol was added dropwise to the reaction system in which the solvent had been removed. After stirring at 90°C for 24 h, methanol and acidic aluminum oxide were added, and the polymerization mixture was obtained by dissolution, filtration, and rotary evaporation. The solution of compound 1 was obtained by flash column chromatography, and the solvent was removed by rotary evaporation to obtain the compound Trt-NH-EG 68 -hPG.

[0156] (3) Synthesis of NH2-EG 68 -hPG 55

[0157] Trt-NH-EG 6-hPG 1.0 g was dissolved in 0.8 mL dichloromethane and 0.8 mL trifluoroacetic acid, and the reaction was stirred in an ice bath for 24 h. The solvent and trifluoroacetic acid were removed by vacuum suction, and the product was frozen and centrifuged in n-hexane for 3 times and dried in a vacuum drying oven overnight to obtain a brown oily liquid NH2-EG 68 -hPG.

[0158] (4) Synthesis of hyperbranched macromonomer MM-19;

[0159] 0.129 mmol Norb-GABA-NHS and 0.071 mmol NH2-EG 68 -hPG was dissolved in 0.8 mL N,N-dimethylformamide and reacted at room temperature for 48 h. The solvent was removed by rotary evaporation, the crude product was dissolved in cold ethanol, and the insoluble matter was removed by filtration. The solvent was removed by rotary evaporation, and the precipitate was obtained by low-temperature centrifugation in n-hexane, and then dried in a vacuum drying oven overnight to obtain a brown oily liquid, which was the hyperbranched macromonomer MM-19.

[0160] (5) Synthesis of bottlebrush polymer BB-20

[0161] 0.250 g MM-19 was dissolved in 1 mL benzene, and anhydrous MM-19 was obtained by vacuum double-tube freezing and water removal. MM-19 was dissolved in 0.1 mL dichloromethane, 0.61 mg Grubbs-III catalyst was added, and the reaction was stirred. After 1 h, a drop of ethyl vinyl ether was added to terminate the reaction, and then the solvent was removed by rotary evaporation. The polymerization mixture was dissolved in 5 mL methanol, and the bottlebrush polymer solution was separated by centrifugation in an ultrafiltration centrifuge tube. The solvent was removed by rotary evaporation to obtain a brown oily liquid, which was the bottlebrush polymer BB-20.

[0162] (6) Synthesis of BB-20-PTX

[0163] 0.050 g of bottlebrush polymer was dissolved in 1.0 mL of N,N-dimethylformamide, 0.077 g of succinic anhydride and 0.5 mg of 4-dimethylaminopyridine were added, and the reaction was stirred at room temperature for 20 h. The solvent was removed by vacuum suction, a small amount of dichloromethane was added, and the insoluble matter was removed by filtration, followed by rotary evaporation to remove the dichloromethane, and centrifugation in ethyl ether to obtain a precipitate. The precipitate was dried in a vacuum drying oven overnight to obtain a brown viscous liquid. 0.100 g of the viscous liquid, 0.046 g of paclitaxel, 0.011 g of N,N'-dicyclohexyl carbodiimide, and 0.001 g of 4-dimethylaminopyridine were dissolved in 0.8 mL of N,N-dimethylformamide, and the reaction was carried out at room temperature for 24 h. The treatment method was the same as before, and a grayish-white solid was obtained by drying, which was the ROMP drug-loaded bottlebrush polymer of hyperbranched polyglycerol.

[0164] Example 10

[0165] A drug-loaded bottlebrush polymer of hyperbranched polyglycerol was synthesized according to the following steps:

[0166] (1) Synthesis of Trt-NH-EG 77 -OH

[0167] 1.0 g of N2H-EG 77 -OH was dissolved in 5 mL of dichloromethane, 0.030 g of triethylamine was added to obtain solution 1. 0.090 g of triphenylmethyl chloride was dissolved in 3 mL of dichloromethane, and the solution was added dropwise to solution 1 under ice bath. After reaction at room temperature for 2.5 h, the solvent was removed by rotary evaporation, and the insoluble matter was removed by filtration after dissolution in toluene, and the crude product was obtained by rotary evaporation. The solution of Trt-NH-EG 77 -OH was obtained by flash column chromatography, and the white solid obtained by rotary evaporation of the solvent was Trt-NH-EG 77 -OH.

[0168] (2) Synthesis of Trt-NH-EG 77 -hPG 55

[0169] 0.060 g of cesium hydroxide monohydrate was dissolved in 30 mL of benzene, and 1.0 g of Trt-NH-EG 77 -OH was added. After stirring at 90°C for 3 h, the solvent was removed by vacuum double-tube suction. 25 mL of glycidol was added to 5.0 g of CaH2, and the mixture was stirred overnight to remove water. Anhydrous glycidol was obtained by distillation under reduced pressure at 60°C and 15 mmHg, and 2.4 mL of anhydrous glycidol was added dropwise to the reaction system after removal of oxygen by freezing. After stirring at 90°C for 24 h, methanol and acidic aluminum oxide were added, the mixture was dissolved and filtered, and rotary evaporation was performed to obtain a polymerization mixture. The solution of compound 1 was obtained by flash column chromatography, and the solvent was removed by rotary evaporation to obtain the compound Trt-NH-EG 77 -hPG.

[0170] (3) Synthesis of NH2-EG 77 -hPG 55

[0171] Trt-NH-EG 77 -hPG 1.0 g was dissolved in 0.8 mL dichloromethane and 0.8 mL trifluoroacetic acid, and the reaction was stirred in an ice bath for 24 h. The solvent and trifluoroacetic acid were removed by vacuum suction, and the product was frozen in n-hexane and centrifuged for 3 times, and dried in a vacuum drying oven overnight to obtain brown oily liquid NH2-EG. 77 -hPG.

[0172] (4) Synthesis of hyperbranched macromonomer MM-21;

[0173] 0.122 mmol Norb-GABA-NHS and 0.068 mmol NH2-EG 77 -hPG was dissolved in 0.8 mL N,N-dimethylformamide and reacted at room temperature for 48 h. The solvent was removed by rotary evaporation, the crude product was dissolved in cold ethanol, and the insoluble matter was removed by filtration. The solvent was removed by rotary evaporation, and the precipitate was obtained by low-temperature centrifugation in n-hexane, and then dried in a vacuum drying oven overnight to obtain brown oily liquid, which was the hyperbranched macromonomer MM-21.

[0174] (5) Synthesis of bottlebrush polymer BB-22

[0175] 0.250 g MM-21 was dissolved in 1 mL benzene, and anhydrous MM-21 was obtained by vacuum double-tube freezing dehydration. MM-21 was dissolved in 0.1 mL dichloromethane, 0.57 mg Grubbs-III catalyst was added, and the reaction was stirred. After 1 h, a drop of ethyl vinyl ether was added to terminate the reaction, and then the solvent was removed by rotary evaporation. The polymerization mixture was dissolved in 5 mL methanol, and the bottlebrush polymer solution was separated by centrifugation in an ultrafiltration centrifuge tube. The solvent was removed by rotary evaporation to obtain brown oily liquid, which was the bottlebrush polymer BB-22.

[0176] (6) Synthesis of BB-22-PTX

[0177] 0.050 g bottlebrush polymer was dissolved in 1.0 mL N,N-dimethylformamide, 0.073 g succinic anhydride and 0.4 mg 4-dimethylaminopyridine were added, and the reaction was stirred at room temperature for 20 h. The solvent was removed by vacuum evaporation, a small amount of dichloromethane was added, and the insoluble matter was removed by filtration, followed by removal of dichloromethane by rotary evaporation, and the precipitate was obtained by centrifugation at low temperature in diethyl ether. The brown viscous liquid was dried in a vacuum drying oven overnight to obtain a brown viscous liquid. 0.100 g of the viscous liquid, 0.045 g of paclitaxel, 0.011 g of N,N'-dicyclohexyl carbodiimide, and 0.001 g of 4-dimethylaminopyridine were dissolved in 0.8 mL of N,N-dimethylformamide, and the reaction was stirred at room temperature for 24 h. The treatment method was the same as before, and a gray-white solid was obtained by drying, which was the ROMP drug-loaded bottlebrush polymer of hyperbranched polyglycerol.

[0178] Example 11

[0179] A drug-loaded bottlebrush polymer of hyperbranched polyglycerol was synthesized according to the following steps:

[0180] (1) Synthesis of Trt-NH-EG 113 -OH

[0181] 1.0 g of N2H-EG 113 -OH was dissolved in 5 mL of dichloromethane, 0.020 g of triethylamine was added to obtain solution 1. 0.061 g of triphenylmethyl chloride was dissolved in 3 mL of dichloromethane, and the solution was added dropwise to solution 1 under ice bath. After stirring at room temperature for 2.5 h, the solvent was removed by rotary evaporation, and the insoluble matter was removed by filtration after dissolving in toluene, and the crude product was obtained by rotary evaporation. The solution of Trt-NH-EG 113 -OH was obtained by flash column chromatography, and the white solid obtained by rotary evaporation of the solvent was Trt-NH-EG 113 -OH.

[0182] (2) Synthesis of Trt-NH-EG 113 -hPG 55

[0183] 0.042 g of cesium hydroxide monohydrate was dissolved in 30 mL of benzene, and 1.0 g of Trt-NH-EG 113 -OH was added. After stirring at 90°C for 3 h, the solvent was removed by vacuum double-tube evaporation. 25 mL of glycidol was added to 5.0 g of CaH2, and the water was removed by stirring overnight. Anhydrous glycidol was obtained by distillation under reduced pressure at 60°C and 15 mmHg, and after removing oxygen by freezing, 1.7 mL of anhydrous glycidol was added dropwise to the reaction system in which the solvent had been removed. After stirring at 90°C for 24 h, methanol and acidic aluminum oxide were added, and the polymerization mixture was obtained by dissolving and filtering and rotary evaporation. The solution of compound 1 was obtained by flash column chromatography, and the solvent was removed by rotary evaporation to obtain the compound Trt-NH-EG 113 -hPG.

[0184] (3) Synthesis of NH2-EG 113 -hPG 55

[0185] Trt-NH-EG 113 -hPG 1.0 g was dissolved in 0.8 mL dichloromethane and 0.8 mL trifluoroacetic acid, and the reaction was stirred in an ice bath for 24 h. The solvent and trifluoroacetic acid were removed by vacuum suction, and the product was frozen in n-hexane and centrifuged for 3 times, and dried in a vacuum drying oven overnight to obtain brown oily liquid NH2-EG. 113 -hPG.

[0186] (4) Synthesis of hyperbranched macromonomer MM-23;

[0187] 0.100 mmol Norb-GABA-NHS and 0.056 mmol NH2-EG 113 -hPG was dissolved in 0.8 mL N,N-dimethylformamide and reacted at room temperature for 48 h. The solvent was removed by rotary evaporation, the crude product was dissolved in cold ethanol, and the insoluble matter was removed by filtration. The solvent was removed by rotary evaporation, and the precipitate was obtained by low-temperature centrifugation in n-hexane, and then dried in a vacuum drying oven overnight to obtain brown oily liquid, which was the hyperbranched macromonomer MM-23.

[0188] (5) Synthesis of bottlebrush polymer BB-24

[0189] 0.250 g MM-23 was dissolved in 1 mL benzene, and anhydrous MM-23 was obtained by vacuum double-tube freezing dehydration. MM-23 was dissolved in 0.1 mL dichloromethane, 0.48 mg Grubbs-III catalyst was added, and the reaction was stirred. After 1 h, a drop of ethyl vinyl ether was added to terminate the reaction, and then the solvent was removed by rotary evaporation. The polymerization mixture was dissolved in 5 mL methanol, and the bottlebrush polymer solution was separated by centrifugation in an ultrafiltration centrifuge tube. The solvent was removed by rotary evaporation to obtain brown oily liquid, which was the bottlebrush polymer BB-24.

[0190] (6) Synthesis of BB-24-PTX

[0191] 0.050 g bottlebrush polymer was dissolved in 1.0 mL of N,N-dimethylformamide, 0.060 g succinic anhydride and 0.4 mg of 4-dimethylaminopyridine were added, and the reaction was stirred at room temperature for 20 h. The solvent was removed by vacuum evaporation, a small amount of dichloromethane was added, and the insoluble material was removed by filtration, followed by removal of dichloromethane by rotary evaporation. The precipitate was obtained by centrifugation at low temperature in diethyl ether. The brown viscous liquid was dried in a vacuum oven overnight to obtain a white solid. 0.100 g of the viscous liquid, 0.040 g of paclitaxel, 0.010 g of N,N'-dicyclohexylcarbodiimide, and 0.001 g of 4-dimethylaminopyridine were dissolved in 0.8 mL of N,N-dimethylformamide, and the reaction was stirred at room temperature for 24 h. The product was obtained by the same procedure as described above, and dried to obtain a white solid, which was the ROMP drug-loaded bottlebrush polymer of hyperbranched polyglycerol.

[0192] Example 12

[0193] A ROMP drug-loaded bottlebrush polymer of hyperbranched polyglycerol was synthesized according to the following steps:

[0194] (1) Synthesis of Trt-NH-EG 227 -OH

[0195] 1.0 g of N2H-EG 227 -OH was dissolved in 5 mL of dichloromethane, and 0.010 g of triethylamine was added to obtain solution 1. 0.031 g of triphenylmethyl chloride was dissolved in 1 mL of dichloromethane, and the solution was added dropwise to solution 1 under ice bath. After the reaction was stirred at room temperature for 2.5 h, the solvent was removed by rotary evaporation, and the insoluble material was removed by filtration after dissolution in toluene. The crude product was obtained by rotary evaporation, and was purified by flash chromatography column to obtain a solution of Trt-NH-EG 227 -OH, and the white solid obtained by rotary evaporation of the solvent was Trt-NH-EG 227 -OH.

[0196] (2) Synthesis of Trt-NH-EG 227 -hPG 55

[0197] 0.021 g of cesium hydroxide monohydrate was dissolved in 30 mL of benzene, and 1.0 g of Trt-NH-EG 227 -OH was added. After stirring at 90 °C for 3 h, the solvent was removed by vacuum double-tube evaporation. 5 mL of glycidol was added to 1.0 g of CaH2, and the mixture was stirred overnight to remove water. Anhydrous glycidol was obtained by distillation under reduced pressure at 60 °C and 15 mmHg, and 0.9 mL of anhydrous glycidol was added dropwise to the reaction system after removal of oxygen by freezing. After stirring at 90 °C for 24 h, methanol and acidic alumina were added, and the mixture was dissolved and filtered to obtain a polymerization mixture. The mixture was purified by flash chromatography column to obtain a solution of compound 1, and the white solid obtained by rotary evaporation of the solvent was Trt-NH-EG 227 -hPG.

[0198] (3) Synthesis of NH2-EG 227 -hPG 55

[0199] Trt-NH-EG 227 -hPG 1.0 g was dissolved in 0.8 mL dichloromethane and 0.8 mL trifluoroacetic acid, and the reaction was stirred in an ice bath for 24 h. The solvent and trifluoroacetic acid were removed by vacuum suction, and the product was frozen in n-hexane and centrifuged for 3 times, and dried in a vacuum drying oven overnight to obtain brown oily liquid NH2-EG. 227 -hPG.

[0200] (4) Synthesis of hyperbranched macromonomer MM-25

[0201] 0.064 mmol Norb-GABA-NHS and 0.036 mmol NH2-EG 227 -hPG was dissolved in 0.8 mL N,N-dimethylformamide and reacted at room temperature for 48 h. The solvent was removed by rotary evaporation, the crude product was dissolved in cold ethanol, and the insoluble matter was removed by filtration. The solvent was removed by rotary evaporation, and the precipitate was obtained by low-temperature centrifugation in n-hexane, and then dried in a vacuum drying oven overnight to obtain brown oily liquid, which was the hyperbranched macromonomer MM-25.

[0202] (5) Synthesis of bottlebrush polymer BB-26

[0203] 0.250 g MM-25 was dissolved in 1 mL benzene, and anhydrous MM-25 was obtained by vacuum double-tube freezing dehydration. MM-25 was dissolved in 0.1 mL dichloromethane, 0.31 mg Grubbs-III catalyst was added, and the reaction was stirred. After 1 h, a drop of ethyl vinyl ether was added to terminate the reaction, and then the solvent was removed by rotary evaporation. The polymerization mixture was dissolved in 5 mL methanol, and the bottlebrush polymer solution was separated by centrifugation in an ultrafiltration centrifuge tube. The solvent was removed by rotary evaporation to obtain brown oily liquid, which was the bottlebrush polymer BB-26.

[0204] (6) Synthesis of BB-26-PTX

[0205] 0.050 g of the bottlebrush polymer was dissolved in 1.0 mL of N,N-dimethylformamide, 0.039 g of succinic anhydride and 0.2 mg of 4-dimethylaminopyridine were added, and the reaction was stirred at room temperature for 20 h. The solvent was removed by vacuum suction, a small amount of dichloromethane was added, and the insoluble matter was removed by filtration, followed by rotary evaporation to remove the dichloromethane, and centrifugation in ethyl ether to obtain a precipitate. The precipitate was dried in a vacuum drying oven overnight to obtain a brown viscous liquid. 0.100 g of the viscous liquid, 0.030 g of paclitaxel, 0.007 g of N,N'-dicyclohexyl carbodiimide, and 0.001 g of 4-dimethylaminopyridine were dissolved in 0.8 mL of N,N-dimethylformamide, and the reaction was carried out at room temperature for 24 h. The treatment method was the same as before, and a grayish-white solid was obtained by drying, which was the ROMP drug-loaded bottlebrush polymer of hyperbranched polyglycerol.

[0206] Example 13

[0207] A drug-loaded bottlebrush polymer of hyperbranched polyglycerol was synthesized according to the following steps:

[0208] (1) Synthesis of Trt-NH-(CH2)6-OH

[0209] 0.5 g of 6-amino-1-hexanol was dissolved in 5 mL of dichloromethane, and 0.432 g of triethylamine was added to obtain solution 1. 1.308 g of triphenylmethyl chloride was dissolved in 3 mL of dichloromethane, and the solution was added dropwise to solution 1 under ice bath. After reaction at room temperature for 2.5 h, the solvent was removed by rotary evaporation, and the insoluble matter was removed by filtration after dissolution in toluene, and the crude product was obtained by rotary evaporation. The solution of Trt-NH-(CH2)6-OH was separated and purified by flash chromatography column, and the solvent was removed by rotary evaporation to obtain white solid, which was Trt-NH-(CH2)6-OH.

[0210] (2) Synthesis of Trt-NH-(CH2)6-hPG 55

[0211] 0.607 g of cesium hydroxide monohydrate was dissolved in 30 mL of benzene, and 1.0 g of Trt-NH-(CH2)6-OH was added. After stirring at 90°C for 3 h, the solvent was removed by vacuum double-tube suction. 50 mL of glycidol was added to 8.0 g of CaH2, and the water was removed by stirring overnight. Anhydrous glycidol was obtained by distillation under reduced pressure at 60°C and 15 mmHg, and 24.0 mL of anhydrous glycidol was added dropwise to the reaction system after removing oxygen by freezing. After stirring at 90°C for 24 h, methanol and acidic aluminum oxide were added, and the polymerization mixture was obtained by dissolution, filtration, and rotary evaporation. The solution of compound 1 was separated and purified by flash chromatography column, and the solvent was removed by rotary evaporation to obtain the compound Trt-NH-(CH2)6-hPG.

[0212] (3) Synthesis of NH2-(CH2)6-hPG 55

[0213] Trt-NH-(CH2)6-hPG 1.0 g was dissolved in 0.8 mL dichloromethane and 0.8 mL trifluoroacetic acid, and the reaction was stirred in an ice bath for 24 h. The solvent and trifluoroacetic acid were removed by vacuum, and the product was frozen in hexane and centrifuged three times and dried in a vacuum oven overnight to give a brown oily liquid, NH2-(CH2)6-hPG.

[0214] (4) Synthesis of hyperbranched macromonomer MM-27;

[0215] 0.220 mmol Norb-GABA-NHS and 0.122 mmol NH2-(CH2)6-hPG were dissolved in 0.8 mL N,N-dimethylformamide and reacted at room temperature for 48 h. The solvent was removed by rotary evaporation, and the crude product was dissolved in cold ethanol and filtered to remove insoluble materials. The solvent was removed by rotary evaporation, and the precipitate was obtained by centrifugation in hexane at low temperature, and then dried in a vacuum oven overnight to give a brown oily liquid, which was hyperbranched macromonomer MM-27.

[0216] (5) Synthesis of bottlebrush polymer BB-28

[0217] 0.250 g MM-27 was dissolved in 3 mL benzene, and anhydrous MM-27 was obtained by vacuum double-tube freezing and dehydration. MM-27 was dissolved in 0.1 mL dichloromethane, and 1.0 mg Grubbs-III catalyst was added and stirred to react. After 1 h, one drop of ethyl vinyl ether was added to terminate the reaction, and then the solvent was removed by rotary evaporation. The polymerization mixture was dissolved in 5 mL methanol, and the bottlebrush polymer solution was separated by centrifugation in an ultrafiltration centrifuge tube, and the solvent was removed by rotary evaporation to give a brown oily liquid, which was bottlebrush polymer BB-28.

[0218] (6) Synthesis of BB-28-PTX

[0219] 0.050 g bottlebrush polymer was dissolved in 1.0 mL N,N-dimethylformamide, and 0.127 g succinic anhydride and 0.8 mg 4-dimethylaminopyridine were added and stirred to react at room temperature for 20 h. The solvent was removed by vacuum, a small amount of dichloromethane was added, and insoluble materials were removed by filtration, and then dichloromethane was removed by rotary evaporation, and the precipitate was obtained by centrifugation in diethyl ether at low temperature. A brown viscous liquid was dried in a vacuum oven overnight. 0.100 g of the viscous liquid, 0.0598 g paclitaxel, 0.0144 g N,N'-dicyclohexyl carbodiimide, and 0.001 g 4-dimethylaminopyridine were dissolved in 0.8 mL N,N-dimethylformamide, and the reaction was carried out at room temperature for 24 h. The product was treated as described above, and a gray-white solid was obtained by drying, which was a ROMP drug-loaded bottlebrush polymer of hyperbranched polyglycerol.

[0220] Comparative Example 1

[0221] Synthesis of MM-4-PTX

[0222] The detailed procedure for preparing MM-4 grafted PTX from Example 1 is as follows:

[0223] 0.200 g of MM-4 was dissolved in 1.6 mL of N,N-dimethylformamide, 0.379 g of succinic anhydride and 0.0023 g of 4-dimethylaminopyridine were added, and the reaction was stirred at room temperature for 20 h. The solvent was removed by vacuum evaporation, a small amount of dichloromethane was added, and the insoluble matter was removed by filtration, followed by removal of dichloromethane by rotary evaporation, and the precipitate was obtained by centrifugation at low temperature in diethyl ether. The brown viscous liquid was dried in a vacuum drying oven overnight to obtain a brown solid, which was MM-4-PTX.

[0224] The average PTX grafting amount was calculated by NMR integration ( Figure 1 ). Each BB-5 bottle brush grafted 330 PTX (7 PTX per MM-4 unit on average). This value means that only 15% of PTX was successfully connected to BB-5, and the grafting reached saturation due to the large steric hindrance of the PTX molecule. About 85% of the -COOH groups on the bottle brush remained intact and could be used to add more functional components. PTX-loaded BB-5 (denoted as BB-5-PTX) still had high water solubility in water (more than 20 mg / mL), and obvious heat was released when dissolved.

[0225] Cell viability assay. The cytotoxicity of the starting H2N-PEG-OH (M.W. = 1000 g / mol), MM-4, BB-5, MM-4-PTX, and BB-5-PTX on various cancer cells (A549, LLC, MDA-MB-231, 4T1, PANC-1, HCT116) and normal cells (BEAS-2B and L02) was studied.

[0226] The table shows the results of the anti-cell proliferation activity of different compounds on different cells (IC 50 , 36 h, n = 3)

[0227]

[0228] In all the cell lines studied, the starting H2N-PEG-OH, hyperbranched polyglycerol macromonomer MM-4 and the common bottlebrush BB-5 did not show any cytotoxicity at the concentration of 4 mg / mL, which is consistent with the common sense that both PEG and hyperbranched polyglycerol are highly biocompatible materials. After loading PTX, all drug-loaded polymers showed inhibition of tumor cell growth. From the net PTX content in different polymers, BB-5-PTX showed 3-20 times higher inhibition of cells than its corresponding monomer MM-4-PTX. This means that the elongated shape of BB-5-PTX significantly improves the efficacy of PTX compared to the spherical shape of MM-4-PTX. To further elucidate the mechanism of this shape effect, we quantitatively studied the endocytosis behavior of Cyanine3-modified MM-4, BB-5, MM-4-PTX and BB-5-PTX using fluorescence confocal microscopy and flow cytometry.

[0229] The endocytosis of MM-4, BB-5, MM-4-PTX and BB-5-PTX was observed by fluorescence microscopy. The average Cy3 labeling amount of all samples was kept at 1 Cy3 molecule per 1 MM-4 unit. The bottlebrush BB-5-PTX loaded with PTX showed stronger fluorescence intensity in cells than MM-4, BB-5 and MM-4-PTX. The intensity of BB-5-PTX was 10 times, 9 times and 8 times that of MM-4-PTX in 2 hours, 6 hours and 12 hours groups, respectively, as shown in Figure 3 . Considering that the Cy3 unit of each BB-5-PTX molecule is 47 times that of MM-4-PTX molecule, the endocytosis molecule number of MM-4-PTX is about 5 times that of BB-5-PTX, but the overall internalized PTX is 8-10 times less. This explains the difference in IC 50 values between MM-4-PTX and BB-5-PTX, and the better tumor cell inhibition is due to the higher internalization of PTX carried by BB-5-PTX. At the same time, the combined effect of the elongated shape of the bottlebrush surface, the negative surface charge, and the discrete hydrophobic sites will enhance the endocytosis of BB-5-PTX by endocytosis, making it a highly efficient drug carrier.

[0230] The mechanism of apoptosis of PTX conjugated to bottlebrush was compared with free PTX by flow cytometry. The C2'-OH group of PTX was converted to an ester bond to conjugate with bottlebrush. Therefore, it is necessary to explore whether the therapeutic effect of PTX changes before and after chemical loading on bottlebrush. Flow cytometry was performed using Annexin V-FITC apoptosis detection kit, which is a widely accepted technique. Annexin V is a calcium-dependent phospholipid binding protein widely distributed in the cytoplasm of eukaryotic cells. It is involved in intracellular signal transduction and can selectively bind to phosphatidylserine (PPS). In living cells, PPS is mainly located on the inner side of the cell membrane. In the early stage of apoptosis, the cell membrane is not affected, but PPS will transfer to the outer side of the cell surface. Using FITC-labeled Annexin V (Annexin V-FITC, showing green fluorescence), flow cytometry can directly detect the externalization of phosphatidylserine. In dead cells, due to the loss of membrane integrity, Annexin V-FITC can enter the cytoplasm and bind to PPS located on the inner side of the cell membrane. On the other hand, propidium iodide (PI, showing red fluorescence) as a small molecule dye can only stain dead cells or late apoptotic cells that have lost membrane integrity. When the cell membrane is still effective, it cannot diffuse into the cell and stain.

[0231] The different conditions for flow cytometry detection of PANC-1 were two net PTX dosing concentrations (15 μΜ and 30 μΜ) and two dosing times (12 h and 24 h). The concentrations of BB-5-PTX and MM-4-PTX were adjusted to achieve the same net PTX concentration. The dosing concentration of the original BB-5 and MM-4 was calculated according to the net polymer content after BB-5-PTX and MM-4-PTX. The same method was applied to MDA-MB-231 cells, except that the net PTX concentration was set to 10 μΜ and 30 μΜ, and the dosing concentration of the polymer was adjusted accordingly. The results are shown in Figure 4

[0232] In Figure 4 each plot, the four quadrants were divided. Q1 included cells with only PI signal (indicating increased membrane permeability) and no Annexin V-FITC signal. The cells in Q2 (upper right) were late apoptotic cells or dead cells, which emitted both PI and Annexin V-FITC signals. Q3 was PTX-induced early apoptotic cells, which emitted only Annexin V-FITC signal and no PI signal. The distribution of the MM-4 group and the BB-5 group was similar to the control group, indicating that both ordinary polymers were non-toxic. The distribution of PTX, BB-5-PTX group and MM-4-PTX group was similar to the PTX group, and the number of cells in Q2 and Q3 quadrants increased sharply Figures 5-8 ​) Q1 is still empty. This means that cell death occurs at the same stage as the PTX group. Therefore, the polymer conjugation of PTX at its C2' position does not change the effect of PTX to induce apoptosis.

[0233] The embodiments described above are part of, but not all, embodiments of the present application. The detailed description of the embodiments of the present application is not intended to limit the scope of the application, but merely to represent selected embodiments of the application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present application.

Claims

1. A process for the preparation of a drug-loaded hyperbranched polyglycerol bottlebrush polymer, characterized in that, The method comprises the following steps: (1) performing water-phase ring-opening metathesis polymerization on the hyperbranched macromonomer by using a Grubbs-III catalyst to obtain a bottlebrush polymer; (2) dissolving the bottlebrush polymer in N,N-dimethylformamide, adding succinic anhydride and 4-dimethylaminopyridine, stirring and reacting at room temperature for 10-30 hours; performing suction filtration, washing and drying to obtain a precipitate; dissolving the precipitate, paclitaxel, N,N'-dicyclohexyl carbodiimide and 4-dimethylaminopyridine in N,N-dimethylformamide, and reacting at room temperature for 20-30 hours; performing suction filtration, washing and drying to obtain a grayish-white solid, which is the drug-loaded bottlebrush polymer of hyperbranched polyglycerol; The molar ratio of the bottlebrush polymer, succinic anhydride and 4-dimethylaminopyridine is 1:5000-30000:0.

01. The structural formula of the hyperbranched macromonomer is as follows: ； In the formula, n=6-227, g, m and k are in the range of 10-60.

2. The production method according to claim 1, characterized by, The synthesis steps of step (1) are as follows: After removing water by freezing, the hyperbranched macromonomer becomes anhydrous hyperbranched macromonomer; the hyperbranched macromonomer is dissolved in dichloromethane, a Grubbs-III catalyst is added, and stirring and reaction are performed at room temperature for 1-2 hours; after the reaction is completed, ethyl vinyl ether is added to terminate the reaction, and then solvent is removed by rotary evaporation to obtain a polymerization mixture; the polymerization mixture is dissolved in methanol, centrifugal separation is performed to obtain a bottlebrush polymer solution, and solvent is removed by rotary evaporation to obtain a brown oily liquid, which is the bottlebrush polymer.

3. The production method according to claim 2, characterized by, The amount of the Grubbs-III catalyst added in step (1) accounts for 0.5%-2% of the mass of the hyperbranched macromonomer.

4. The method of claim 1, wherein, In step (2), the molar ratio of the precipitate, paclitaxel, N,N'-dicyclohexyl carbodiimide and 4-dimethylaminopyridine is 1:300-6000:300-6000:30-600.

5. A hyperbranched polyglycerol drug-loaded bottlebrush polymer, characterized in that, The drug-loaded bottlebrush polymer of hyperbranched polyglycerol is prepared by any one of the methods of claims 1-4.

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