Synthesis of amphiphilic polymer and preparation method of blank micelles

Abstract

The application provides a synthesis of an amphiphilic polymer and a preparation method of a blank micelle. The preparation method of the amphiphilic polymer comprises the following steps: firstly, obtaining a linear polypropylene imine main chain through a cyclization reaction, a cation ring-opening polymerization and a hydrolysis reaction; secondly, modifying tert-butyl acrylate and octadecyl acrylate on the polymer main chain, and then removing the tert-butyl under an acidic condition to obtain the amphiphilic polymer. Then, the amphiphilic polymer is dissolved in a good solvent, deionized water is added dropwise into the good solvent, and the good solvent is dialyzed in the deionized water for a period of time to obtain the blank micelle. The micelle prepared by the application has good size stability in an aqueous solution.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of polymer science, and particularly relates to a synthesis of an amphiphilic polymer and a method for self-assembly of the polymer into a blank micelle. BACKGROUND

[0002] Cancer is one of the major diseases that seriously endanger human life and health. According to the World Health Organization (WHO) 2020 World Cancer Report, one sixth of the annual death toll worldwide is due to cancer, and about 9.96 million people died of cancer worldwide in 2020. The report predicts that the number of cancer cases worldwide may further increase in the next 20 years. At present, the traditional treatment methods for cancer are mainly surgery, radiotherapy and chemotherapy. Surgery is mainly for solid tumors, and it is difficult to deal with metastatic malignant tumors. Chemotherapy can be applied to more types of tumor treatment, but it lacks targeting and is toxic to normal cells while killing tumor cells, so there is an urgent need for a solution that can reduce the toxic side effects of chemotherapy drugs while not affecting their efficacy.

[0003] Nanodrug delivery systems can achieve drug accumulation in tumor sites and reduce the content of drugs in normal tissues through the size advantage of high permeability and long retention effect (EPR effect), thus effectively reducing the toxic side effects of chemotherapy drugs, and have received widespread attention. However, traditional nanodrug delivery systems have poor stability, and the size gradually changes with increasing storage time, making it difficult to exist stably in the blood. SUMMARY

[0004] The purpose of the present application is to provide a synthesis of an amphiphilic polymer and a method for preparing a blank micelle, to solve the problem of poor stability of nanodrug delivery systems, and to prepare an amphiphilic polymer micelle containing zwitterions as hydrophilic segments, which is expected to improve the stability of nanodrug delivery systems. The present application uses zwitterionic polymers with equal positive and negative charges as hydrophilic segments, which have strong binding capacity with water molecules through ionic solvation to resist the protein corona barrier in the blood. It is a better material than traditional anti-protein adsorption material polyethylene glycol (PEG), which can enhance the stability of nanodrug delivery systems.

[0005] To achieve the above purpose, the present application provides a preparation method of an amphiphilic polymer, comprising the following steps:

[0006] 1) Synthesizing monomer 2-methyloxazine from acetonitrile and 3-amino-1-propanol in the presence of a catalyst;

[0007] Preferably, acetonitrile and 3-amino-1-propanol are reacted at 80-115℃ for 24-72h in the presence of a catalyst, and 120-130℃ fraction is collected by normal pressure distillation to obtain monomer 2-methyloxazine.

[0008] Preferably, the catalyst is zinc acetate or zinc chloride or zinc acetate dihydrate, and the molar ratio of acetonitrile to catalyst is 1:0.01-1:0.05, preferably 1:0.02.

[0009] Preferably, the molar ratio of acetonitrile to 3-amino-1-propanol is 1:0.9-1:1.1, preferably 1:1.1.

[0010] Preferably, the reaction temperature is 95-105°C, and the reaction time is 40-48h.

[0011] Specifically, acetonitrile and zinc acetate are added to a three-necked flask, heated to 80-115°C, preferably 95-105°C, and after acetonitrile reflux, 3-amino-1-propanol is slowly added dropwise using a constant pressure dropping funnel, and the reaction is carried out for 24-72h, preferably 40-48h, to obtain 2-methyloxazoline (MeOZI) crude product. The crude product is subjected to atmospheric distillation, and the 120-130°C fraction is collected to obtain colorless transparent liquid as purified 2-methyloxazoline.

[0012] 2) cationic ring-opening polymerization of 2-methyloxazoline monomer to obtain polymethyl oxazoline;

[0013] Preferably, the cationic ring-opening polymerization (CROP) of 2-methyloxazoline (MeOZI) monomer and initiator is carried out in a microwave reactor at 140-150°C for 30-60min after water removal treatment.

[0014] Preferably, the initiator is at least one of methyl-p-toluenesulfonate (MeOTs), methyl trifluoromethanesulfonate, and acetyl chloride.

[0015] Preferably, the molar ratio of monomer MeOZI to initiator is 40:1-175:1, preferably 88:1-100:1.

[0016] Preferably, the reaction solvent is acetonitrile.

[0017] Preferably, 2-methyloxazoline monomer is subjected to water removal using calcium hydride, and the initiator is subjected to water removal using molecular sieves.

[0018] Preferably, 1-2M KOH in methanol is added at 0-5°C, the molar ratio of KOH to initiator is 1:1-2:1, preferably 1:1, and the mixture is stirred at room temperature for 12-24h to terminate polymerization.

[0019] Specifically, first, monomer MeOZI and initiator are pre-treated. MeOZI is dehydrated: calcium hydride is added to MeOZI, and normal pressure distillation is performed to collect a fraction at 120-130°C; wherein the amount of calcium hydride is 5%-10% of the mass of MeOZI. The initiator is dehydrated: molecular sieves are activated in a muffle furnace at 400-500°C for 1-3h, and the activated molecular sieves are placed in the initiator sample and treated at 30-40°C for 24-48h. MeOZI cationic ring-opening polymerization: MeOZI and MeOTs are dissolved in acetonitrile and added to a Biotage microwave vial, and the solution is reacted in a microwave reactor at 140°C for 50min, and the solution changes from colorless to yellow. Thereafter, 1-2M KOH in methanol is added at 0-5°C, and the solution is stirred at room temperature for 12-24h to terminate the polymerization. The solvent is removed by rotary evaporation, and the residual polymer is dissolved in chloroform and precipitated in cold n-hexane for 3-5 times, and the supernatant is removed by centrifugation at 3000-8000rpm, and the product is dried in vacuum at 40°C for 48h to obtain polymethyl oxazine (PMeOZI).

[0020] 3) Hydrolysis of PMeOZI under acidic conditions to obtain linear polypropylene imine (lPPI).

[0021] Preferably, the concentration of hydrogen ions before hydrolysis is 1M-10M, preferably 3.4-4.4M; hydrochloric acid is used to provide the acidic conditions.

[0022] Preferably, the temperature of the hydrolysis is 110-120°C, and the time of the hydrolysis is 6-72h, preferably 18-36h.

[0023] Specifically, PMeOZI is dissolved in hot water at 80-100°C, heated to 110-120°C and refluxed for 15-20min, and then concentrated hydrochloric acid is added for reaction. The reaction time is 6-72h, preferably 18-36h, and the concentration of hydrogen ions before hydrolysis is 1M-10M, preferably 3.4-4.4M. The solvent is removed by rotary evaporation, and the residual solid is dissolved in hot water and neutralized with NaOH solution to pH 9-10, and a large amount of light yellow precipitate is precipitated. Centrifugation is performed at 3000-8000rpm, the supernatant is discarded, and the operation is repeated 3-5 times, and the product is dried in vacuum at 40-65°C for 24-48h to obtain linear polypropylene imine (lPPI).

[0024] 4) Grafting of t-butyl acrylate (tBA) and octadecyl acrylate (ODA) onto the lPPI backbone by Michael addition reaction to obtain amphiphilic polymer precursor (PPI-tBA-ODA).

[0025] Preferably, the molar ratio of tBA to ODA is 1:2.0-14.6:1, preferably 2.9:1-5.8:1.

[0026] Preferably, the molar ratio of the PPI secondary amine group to the sum of tBA and ODA is 1:0.9-1:1.1, preferably 1:1.

[0027] Preferably, the reaction temperature is 35-45℃, and the reaction time is 24-48h.

[0028] Preferably, the reaction solvent is a mixed solvent of ethanol and tetrahydrofuran, and the volume ratio of ethanol to tetrahydrofuran is 1:1-10:1, preferably 2:1-5:1.

[0029] Preferably, PPI and tBA are dissolved in ethanol, and then a THF solution of ODA is added, and mixed at 35-45℃ for 24-48h to obtain the amphiphilic polymer precursor (PPI-tBA-ODA).

[0030] Specifically, PPI and tBA are dissolved in ethanol, and then a THF solution of ODA is added, and mixed at 35-45℃ for 24-48h, rotary evaporation into a concentrated solution, precipitated in the corresponding precipitant for 3-5 times, centrifuged to remove the supernatant at 3000rpm-8000rpm, and vacuum dried at 40℃ for 48h. The precipitant is methanol or water or n-hexane or diethyl ether, preferably methanol or water.

[0031] 5) The amphiphilic polymer precursor is hydrolyzed under acidic conditions to remove the tert-butyl group to obtain the amphiphilic polymer.

[0032] Preferably, PPI-tBA-ODA is dissolved in a mixed solution of acid and corresponding solvent to dissolve the polymer, stirred at 35-45℃ for 2-6h, precipitated in diethyl ether, centrifuged to remove the supernatant, and dried to obtain the deprotected PPI-tBA-ODA, which is the amphiphilic polymer.

[0033] Preferably, the acid is acetic acid or hydroxyacetic acid or trifluoroacetic acid, and the solvent is n-hexane or chloroform or dichloromethane or tetrahydrofuran or acetone, preferably n-hexane or chloroform.

[0034] Preferably, the volume ratio of acid to solvent in the mixed solution of acid and solvent is 1:1-1:2.

[0035] Specifically, PPI-tBA-ODA is dissolved in a mixed solution of acid and corresponding solvent to dissolve the polymer, stirred at 35-45℃ for 2-6h, precipitated in diethyl ether for 3-5 times, centrifuged to remove the supernatant at 3000-8000rpm, and deprotected by vacuum drying at 30-40℃ for 24-48h.

[0036] An amphiphilic polymer blank micelle, comprising the above-mentioned amphiphilic polymer.

[0037] A preparation method of an amphiphilic polymer blank micelle, which is self-assembled from the above-mentioned amphiphilic polymer in an aqueous solution.

[0038] Preferably, the amphiphilic polymer is dissolved in a good solvent under fast stirring, and deionized water is slowly added dropwise to form the blank micelles.

[0039] Preferably, the good solvent is tetrahydrofuran (THF) or acetonitrile or ethanol.

[0040] Preferably, the ratio of the amphiphilic polymer, the good solvent and the deionized water is 10-20 mg: 1-2 mL: 4-9 mL.

[0041] Preferably, the amphiphilic polymer is dissolved in a good solvent, a small amount of deionized water is first added dropwise to promote self-assembly of the polymer, and then a large amount of deionized water is added to fix the assembly; wherein the volume ratio of the small amount of water to the large amount of water is 0.2-0.5:4-8, for example, the amount of the small amount of water is 200-500 μL, and the amount of the large amount of water is 4-8 mL.

[0042] Preferably, the stirring speed is 1000-1200 rpm, and the stirring time is 2-4 h.

[0043] Preferably, the speed of dropwise addition is 1-5 mL / min, preferably 1-2 mL / min.

[0044] Preferably, after the polymer self-assembles into blank micelles, the solution is dialyzed in deionized water for 12-72 h, preferably 24-48 h, and the Mw of the dialysis is 3500-5000 Da.

[0045] Preferably, the water is changed every 4-6 h during dialysis.

[0046] Preferably, after dialysis, the micelle solution is diluted to 0.5-1 mg / mL to obtain a blank micelle solution.

[0047] Specifically, the amphiphilic polymer (10 mg) is dissolved in a good solvent (1-2 mL), and deionized water (200-500 μL) is added dropwise at a speed of 1-5 mL / min, preferably 1-2 mL / min, under stirring at 1000-1200 rpm for 1-2 h to promote self-assembly of the polymer, and then a large amount of water (4-8 mL) is added dropwise to fix the assembly, the solution is transferred to a dialysis bag (Mw = 3500-5000 Da), and dialyzed in deionized water for 12-72 h, preferably 24-48 h, and the water is changed every 4-6 h to remove the organic solvent, and after dialysis, the mother liquor is diluted to 0.5-1 mg / mL to obtain a blank micelle solution.

[0048] The application prepares an amphiphilic polymer, which has hydrophilic zwitterions to improve water solubility and stability of the carrier, and hydrophobic octadecyl groups to load hydrophobic drugs. After the polymer is dissolved in a good solvent, water is slowly added to the solution, and the hydrophobic segments will gather in the inside to form a hydrophobic core, and the hydrophilic segments will be exposed to the outside to form a hydrophilic shell. The micelle structure after self-assembly can be used to load hydrophobic drugs, and the solubility of the hydrophobic drugs in aqueous solution is enhanced.

[0049] Advantages:

[0050] 1) The hydrophilic zwitterion structure has equal positive and negative charges, and can have greater binding capacity with water molecules through ionic solvation, thereby enhancing the hydrophilicity of the polymer. The micelle after self-assembly has a hydrophilic zwitterion shell, and has good stability in aqueous solution.

[0051] 2) The hydrophobic octadecyl structure can load hydrophobic drugs, and during self-assembly, the hydrophobic drugs are wrapped in the core together with the octadecyl structure. This kind of amphiphilic polymer can increase the solubility of the hydrophobic drugs in aqueous solution.

[0052] 3) The size of the drug-loaded micelle after self-assembly is about 200-300 nm, which meets the standard of EPR effect, and is a suitable nano drug delivery system. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 NMR spectrum of monomer 2-methyl oxazine.

[0054] Figure 2 NMR spectrum of polymethyl oxazine (PMeOZI).

[0055] Figure 3 NMR spectrum of linear polypropylene imine (lPPI).

[0056] Figure 4 NMR spectrum of four amphiphilic polymer precursors (PPI-tBA-ODA) with different hydrophilic and hydrophobic ratios, wherein (a)-(d) correspond to Examples 1-4, respectively.

[0057] Figure 5 NMR spectrum of four amphiphilic polymers (PPICB-ODA) with different hydrophilic and hydrophobic ratios, wherein (a)-(d) correspond to Examples 1-4, respectively.

[0058] Figure 6 Particle size (a) and zeta potential (b) of four blank micelles, wherein 1-4 correspond to Examples 1-4, respectively.

[0059] Figure 7: Size stability of four blank micelles solutions within 14 days, wherein (a)-(d) correspond to Examples 1-4, respectively.

[0060] Figure 8 : UV spectra of blank micelles and drug-loaded micelles. DETAILED DESCRIPTION

[0061] The present application provides a kind of amphiphilic polymer micelles and its preparation method. The method comprises the following steps: first, linear polypropylene imine is synthesized, then two kinds of acrylic ester, tert-butyl acrylate and octadecyl acrylate, are grafted on the main chain, and the tert-butyl group is removed under acidic conditions to obtain amphiphilic polymer. The polymer is dissolved in a good solvent, and water is slowly added to obtain blank micelles.

[0062] The following specific examples are further illustrations of the present application and should not be construed as limiting the application in any way.

[0063] Example 1

[0064] 1) Add anhydrous acetonitrile (596 mmol, 31.2 mL), zinc acetate dihydrate (11.93 mmol, 2.62 g) into a 250 mL three-necked flask, heat to 80°C, and then slowly add 3-amino-1-propanol (657 mmol, 50 mL) into the flask using a constant pressure dropping funnel. After refluxing for 40 h, 2-methyloxaziridine (MeOZI) is obtained. The crude product is subjected to atmospheric distillation, and the fraction collected at 120-130°C is a colorless transparent liquid, MeOZI, with a yield of 35%. The structure of the monomer MeOZI is determined by Figure 1 The structure of the monomer MeOZI is determined by

[0065] 2) First, monomer MeOZI and initiator methyl-p-toluenesulfonate (MeOTs) were pre-dried. MeOZI was dried by adding 5% mass fraction of calcium hydride, and distilling at normal pressure to collect the fraction of 120-130 °C. MeOTs was dried by activating 3A molecular sieves in a muffle furnace at 400 °C for 3 h, and then putting the activated molecular sieves into a 50 mL beaker containing 15 g of sample, so that the molecular sieves covered the bottom of the beaker. The beaker was treated at 30 °C for 24 h and then used. Cationic ring-opening polymerization of MeOZI (polymerization degree 100): MeOZI (50 mmol, 4.7 mL) and MeOTs (0.5 mmol, 75.4 μL) were mixed in a 20 mL Biotage microwave vial with super-dry acetonitrile (7.7 mL). The solution was reacted in a microwave reactor at 140 °C for 50 min, and the solution changed from colorless to yellow. Then, 1 M KOH in methanol (0.25 mL) was added at 0 °C, and the solution was stirred at room temperature overnight to terminate the polymerization. The solvent was removed by rotary evaporation, and the residual polymer was dissolved in chloroform (5 mL) and precipitated in cold n-hexane (45 mL) for 3 times, and dried at 40 °C under vacuum for 48 h to obtain a light yellow sticky solid with a yield of 95%. The structure of PMeOZI was determined by Figure 2 PMeOZI structure was determined.

[0066] 3) PMeOZI (22.67 mmol, 2.2469 g) was dissolved in hot water (17 mL) at 100 °C, and then 37% hydrochloric acid (30 mL) was added after heating to reflux at 120 °C for 15 min. The reaction was performed for 36 h. The solvent was removed by rotary evaporation, and the residual solid was dissolved in hot water (77 mL) and neutralized to pH 9 with 0.1 M NaOH solution. A large amount of light yellow precipitate was obtained. The solution was centrifuged at 3000 rpm, and the supernatant was discarded. The operation was repeated 3 times, and the product was dried at 65 °C under vacuum for 48 h to obtain an orange-yellow crystalline solid polypropylenimine (PPI) with a yield of 98%. The structure of PPI was determined by Figure 3 PPI structure was determined.

[0067] 4) PPI (300 mg, 5.25 mmol) was dissolved in ethanol (15 mL) with tBA (249 μL), and then a THF solution (5 mL) of ODA (1136 mg) was added. The mixture was stirred at 35 °C for 48 h, and then concentrated to 5 mL by rotary evaporation. The solution was precipitated in methanol (45 mL) for 3 times, and the supernatant was removed by centrifugation. The product was dried at 40 °C under vacuum for 48 h. The structure of PPI-tBA-ODA was determined by Figure 4 (a) PPI-tBA-ODA structure was determined.

[0068] 5) PPI-tBA-ODA (252 mg) was dissolved in a mixture of TFA / n-hexane (1:1) (4 mL) to dissolve the polymer, and then stirred at 35 °C for 6 h. The solution was precipitated in ether for 3 times, and the supernatant was removed by centrifugation. The product was dried at 40 °C under vacuum for 48 h. The structure of PPI-tBA-ODA was determined by Figure 5(a) The PPICB-ODA structure was determined.

[0069] 6) Dissolve the amphiphilic polymer (20 mg) in a good solvent, THF (2 mL). Add 2 mL of deionized water dropwise at 1 mL / min at 1000 rpm, stirring for 2 h to promote polymer self-assembly. Then, add 8 mL of deionized water dropwise at 1 mL / min to fix the assemblies, stirring for 2 h. Transfer the solution to a dialysis bag (Mw = 3500 Da) and dialyze for 24 h, changing the water every 6 h to remove the organic solvent. After dialysis, bring the mother liquor to a final volume of 1 mg / mL, filter through a 0.45 μm filter membrane, and test the particle size and zeta potential. Simultaneously, test the dimensional stability of the obtained blank micelles after storage for different times. After 14 days of storage, the micelle size increased slightly, but the stability was generally moderate.

[0070] Example 2

[0071] The difference between this embodiment and Example 1 lies in the amount of tert-butyl acrylate and octadecyl acrylate added: 555 μL of tert-butyl acrylate and 436 mg of octadecyl acrylate. Figure 4 (b) The PPI-tBA-ODA structure was determined. Figure 5 (b) The PPICB-ODA structure was determined. Time stability test results are as follows: Figure 2 As shown. Compared to Example 1, the blank micelle size remained stable after 14 days of storage.

[0072] Example 3

[0073] The difference between this embodiment and Example 1 lies in the different amounts of tert-butyl acrylate and octadecyl acrylate, and the different precipitant. The tert-butyl acrylate is 638 μL, the octadecyl acrylate is 249 mg, and the precipitant is water. Figure 4 (c) The PPI-tBA-ODA structure was determined. Figure 5 (c) The PPICB-ODA structure was determined. Time stability test results are as follows: Figure 2 As shown. Compared to Example 1, the blank micelle size remained stable after 14 days of storage.

[0074] Example 4

[0075] The difference between this embodiment and Example 1 lies in the different amounts of tert-butyl acrylate and octadecyl acrylate, and the different precipitant. In this example, 700 μL of tert-butyl acrylate and 109 mg of octadecyl acrylate are used, with water as the precipitant. Figure 4 (d) The PPI-tBA-ODA structure was determined. Figure 5 (d) The PPICB-ODA structure was determined. Time stability test results are as follows:Figure 2 The blank micelles size decreased and the stability decreased after 14 days of storage compared to Example 3.

[0076] Example 5

[0077] 1) Dry acetonitrile (596 mmol, 31.2 mL), zinc acetate dihydrate (11.93 mmol, 2.62 g) were added into a 250 mL three-necked flask, heated to 105 °C, acetonitrile refluxed, 3-amino-1-propanol (657 mmol, 50 mL) was added slowly into the flask using a constant pressure dropping funnel, the reaction was carried out for 24 h to give 2-methyloxazoline (MeOZI). The crude product was distilled at atmospheric pressure, the fraction of 120-130 °C was collected to give colorless transparent liquid MeOZI with a yield of 24%.

[0078] 2) First, the monomer MeOZI and the initiator methyl-p-toluenesulfonate (MeOTs) were pre-treated by water removal. MeOZI water removal: 5% mass fraction of calcium hydride was added into MeOZI, distilled at atmospheric pressure, the fraction of 120-130 °C was collected. Initiator methyl-p-toluenesulfonate (MeOTs) water removal: 3A molecular sieves were activated in a muffle furnace at 400 °C for 3 h, the activated molecular sieves were put into a 50 mL beaker with 15 g of sample, the molecular sieves were spread on the bottom of the beaker, and the beaker was treated at 30 °C for 24 h before use. Cationic ring-opening polymerization of MeOZI (polymerization degree 40): MeOZI (50 mmol, 4.7 mL) and MeOTs (1.25 mmol, 171.4 μL), dry acetonitrile (7.7 mL) were mixed in a 20 mL Biotage microwave vial, the solution was reacted in a microwave reactor at 140 °C for 50 min, the solution changed from colorless to yellow. After that, 1 M KOH in methanol (0.25 mL) was added at 0 °C, the solution was stirred at room temperature overnight to terminate the polymerization. The solvent was removed by rotary evaporation, the residual polymer was dissolved in chloroform (5 mL), precipitated and purified in cold n-hexane (45 mL) for 3 times, and dried at 40 °C under vacuum for 48 h to give a light yellow sticky solid with a yield of 98%.

[0079] 3) PMeOZI (22.67 mmol, 2.2469 g) was dissolved in hot water (17 mL) at 100 °C, 37% hydrochloric acid (6.8 mL) was added after heating to reflux at 120 °C for 15 min, and the reaction was carried out for 6 h. The solvent was removed by rotary evaporation, the residual solid was re-dissolved in hot water (77 mL), and the solution was neutralized to pH 9 using 0.1 M NaOH solution, and a large amount of light yellow precipitate was precipitated. Centrifugation at 3000 rpm, the supernatant was discarded, and the operation was repeated 3 times, and the product was dried at 65 °C under vacuum for 48 h to give orange-yellow crystalline solid polypropylenimine (PPI) with a yield of 21%.

[0080] 4) PPI (300 mg, 5.25 mmol) was dissolved in ethanol (15 mL) with tBA (249 μL) and ODA (1136 mg) in THF (5 mL) was added and mixed at 35 °C for 48 h. The solution was concentrated to 5 mL by rotary evaporation and precipitated in n-hexane (45 mL) for 3 times to get a small amount of product.

[0081] 5) PPI-tBA-ODA (252 mg) was dissolved in a mixture of acetic acid and chloroform (1 : 1) (4 mL) to dissolve the polymer, stirred at 35 °C for 6 h, precipitated in ether for 3-5 times, centrifuged to remove the supernatant, and dried at 40 °C under vacuum for 48 h.

[0082] 6) Amphiphilic polymer (20 mg) was dissolved in a good solvent acetonitrile (2 mL) and dropped into deionized water (2 mL) at 1000 rpm with a speed of 2 mL / min, stirred for 2 h to promote self-assembly of the polymer, and then deionized water (8 mL) was dropped at a speed of 1 mL / min to fix the assembly, stirred for 2 h, and the solution was transferred to a dialysis bag (Mw=3500 Da), dialyzed for 12 h, and the water was changed every 6 h to remove the organic solvent. After dialysis, the mother liquor was diluted to 1 mg / mL, filtered through a 0.45 μm filter membrane to obtain a blank micelle solution.

[0083] Example 6

[0084] 1) Anhydrous acetonitrile (596 mmol, 31.2 mL) and zinc acetate dihydrate (11.93 mmol, 2.62 g) were added to a 250 mL three-necked flask and heated to 115 °C. After acetonitrile refluxed, 3-amino-1-propanol (657 mmol, 50 mL) was slowly dropped into the flask using a constant pressure dropping funnel. The reaction was carried out for 72 h to obtain 2-methyloxazine (MeOZI). The crude product was subjected to atmospheric distillation, and the fraction of 120-130 °C was collected to obtain colorless transparent liquid MeOZI with a yield of 12%.

[0085] 2) First, monomer MeOZI and initiator methyl-p-toluenesulfonate (MeOTs) were pre-dried. MeOZI was dried by adding 5% mass fraction of calcium hydride to MeOZI, and normal pressure distillation was performed to collect the fraction at 120-130 °C. The initiator methyl-p-toluenesulfonate (MeOTs) was dried by activating 3A molecular sieves in a muffle furnace at 400 °C for 3 h, and then the activated molecular sieves were put into a 50 mL small beaker containing 15 g of sample, so that the molecular sieves covered the bottom of the beaker. The beaker was treated at 30 °C for 24 h and then used. Cationic ring-opening polymerization of MeOZI (polymerization degree 175): MeOZI (50 mmol, 4.7 mL) and MeOTs (0.28 mmol, 43.1 μL) were mixed in a 20 mL Biotage microwave vial with super-dry acetonitrile (7.7 mL). The solution was reacted in a microwave reactor at 140 °C for 50 min, and the solution changed from colorless to yellow. Then, 1 M KOH in methanol (0.25 mL) was added at 0 °C, and the solution was stirred at room temperature overnight to terminate the polymerization. The solvent was removed by rotary evaporation, and the residual polymer was dissolved in chloroform (5 mL) and precipitated in cold n-hexane (45 mL) for 3 times, and then dried in vacuum at 40 °C for 48 h to obtain a light yellow sticky solid with a yield of 98%.

[0086] 3) PMeOZI (22.67 mmol, 2.2469 g) was dissolved in hot water (17 mL), heated to reflux for 15 min, and then 37% hydrochloric acid (68 mL) was added, and the reaction was performed for 72 h. The solvent was removed by rotary evaporation, and the residual solid was re-dissolved in hot water (77 mL) and neutralized to pH 9-10 with NaOH solution, and a large amount of light yellow precipitate was separated out. The 3000-5000 ppm was centrifuged, and the supernatant was discarded, and the operation was repeated 3 times, and then dried in vacuum at 65 °C for 48 h to obtain orange-yellow crystalline solid polypropylene imine (PPI) with a yield of 98%. The structure of lPPI was determined by Figure 3

[0087] 4) PPI (300 mg, 5.25 mmol) was dissolved in ethanol (15 mL) with tBA (249 μL), and then a THF solution (5 mL) of ODA (1136 mg) was added, and the mixture was mixed at 35 °C for 48 h, and then concentrated to a solution (5 mL) by rotary evaporation, and then precipitated in ether (45 mL) for 3 times to obtain a small amount of product.

[0088] 5) PPI-tBA-ODA (252 mg) was dissolved in a mixed solution of glycolic acid / dichloromethane (1:1) (4 mL) to dissolve the polymer, and then stirred at 35 °C for 6 h, and then precipitated in ether for 3 times, and then the supernatant was removed by centrifugation, and then dried in vacuum at 40 °C for 48 h.

[0089] ​6) The amphiphilic polymer (20 mg) was dissolved in a good solvent, ethanol (2 mL), and was dropped into deionized water (2 mL) at a speed of 5 mL / min under 1000 rpm, and was stirred for 2 h to promote the self-assembly of the polymer, and then was dropped into deionized water (8 mL) at a speed of 1 mL / min to fix the assembly, and was stirred for 2 h, and then was transferred into a dialysis bag (Mw = 3500 Da), and was dialyzed for 72 h, and the water was changed every 6 h to remove the organic solvent, and after dialysis, the mother liquor was diluted to 1 mg / mL, and was filtered through a 0.45 μm filter to obtain a blank micelle solution.

[0090] Example 7

[0091] The difference between this example and Example 1 is that the specific procedure of Step 6) is as follows: the amphiphilic polymer (20 mg) and docetaxel (3 mg) were dissolved in a good solvent, THF (2 mL), and were dropped into deionized water (2 mL) at a speed of 1 mL / min under 1000 rpm, and were stirred for 2 h to promote the self-assembly of the polymer, and then were dropped into deionized water (8 mL) at a speed of 1 mL / min to fix the assembly, and were stirred for 2 h, and then were transferred into a dialysis bag (Mw = 3500 Da), and were dialyzed for 24 h, and the water was changed every 6 h to remove the organic solvent, and after dialysis, the mother liquor was diluted to 1 mg / mL, and was filtered through a 0.45 μm filter to obtain a drug-loaded micelle solution. The ultraviolet spectra of the blank micelles and the drug-loaded micelles are shown in Figure 8 Fig. 2, and the absorbance of the drug-loaded micelles at 230 nm is enhanced, which proves that docetaxel is loaded in the micelles.

Claims

1. A method for preparing an amphiphilic polymer, characterized by: The method comprises the following steps: 1) synthesizing monomer 2-methyloxazoline by using acetonitrile and 3-amino-1-propanol in the presence of a catalyst; 2) cationic ring-opening polymerization of 2-methyloxazoline monomer to obtain polymethyl oxazoline; 3) hydrolysis of polymethyl oxazoline under acidic conditions to obtain linear polypropylenimine; 4) grafting t-butyl acrylate and octadecyl acrylate on the polypropylenimine backbone to obtain an amphiphilic polymer precursor; 5) hydrolysis of the amphiphilic polymer precursor under acidic conditions to obtain an amphiphilic polymer.

2. The method for preparing an amphiphilic polymer according to claim 1, wherein: In step 1), the reaction temperature is 80-115℃, the reaction time is 24-72h, and the 120-130℃ fraction is collected by normal pressure distillation after the reaction; the molar ratio of acetonitrile to 3-amino-1-propanol is 1:0.9-1:1.1; the catalyst is zinc acetate or zinc chloride or zinc acetate dihydrate, and the molar ratio of acetonitrile to catalyst is 1:0.01-1:0.

05.

3. The method for preparing an amphiphilic polymer according to claim 1, wherein: In step 2), the 2-methyloxazoline monomer and the initiator are treated to remove water, and then cationic ring-opening polymerization is carried out in a microwave reactor at 140-150℃ for 30-60min to obtain polymethyl oxazoline.

4. The method for preparing an amphiphilic polymer according to claim 3, wherein: In step 2), the initiator is at least one of methyl p-toluenesulfonate, methyl trifluoromethanesulfonate, and acetyl chloride, the molar ratio of 2-methyloxazoline monomer to initiator is 40:1-175:1, the reaction solvent is acetonitrile, and calcium hydride is used to remove water from the 2-methyloxazoline monomer, and molecular sieves are used to remove water from the initiator.

5. The method for preparing an amphiphilic polymer according to claim 1, wherein: In step 3), the hydrogen ion concentration before hydrolysis is 1-10M, the reaction temperature is 110-120℃, and the reaction time is 6-72h.

6. The method for preparing an amphiphilic polymer according to claim 1, wherein: In step 4), the molar ratio of t-butyl acrylate to octadecyl acrylate is 1:2.0-14.6:1, the molar ratio of the secondary amine group of the polypropylenimine to the sum of t-butyl acrylate and octadecyl acrylate is 1:0.9-1:1.1, the reaction temperature is 35-45℃, and the reaction time is 24-48h; the reaction solvent is a mixed solvent of ethanol and tetrahydrofuran, and the volume ratio of ethanol to tetrahydrofuran is 1:1-10:1; the precipitant used is water or methanol or diethyl ether or n-hexane.

7. The method for preparing an amphiphilic polymer according to claim 1, wherein: In step 5), the amphiphilic polymer precursor is dissolved in a mixed solution of acid and solvent, stirred at 35-45℃ for 2-6h, precipitated in diethyl ether, centrifuged to remove the supernatant, and dried to obtain the amphiphilic polymer; wherein the acid is acetic acid or hydroxyacetic acid or trifluoroacetic acid, the solvent is n-hexane or chloroform or dichloromethane, and the volume ratio of acid to solvent in the mixed solution of acid and solvent is 1:1-1:

2.

8. The amphiphilic polymer prepared by the preparation method of any one of claims 1-7.

9. An amphiphilic polymer blank micelle comprising the amphiphilic polymer of claim 8.

10. The method for preparing an amphiphilic polymer blank micelle according to claim 9, wherein the hydrophobic polymer is a polymer having a hydrophobic group in the molecule. The amphiphilic polymer blank micelle is self-assembled from the amphiphilic polymer in an aqueous solution.

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