A method for preparing a polymer micelle loaded with a hydrophobic drug

Abstract

The application provides a preparation method of a high-molecular drug-loaded micelle loaded with a hydrophobic drug. The preparation method is as follows: an amphiphilic polymer and a hydrophobic drug are dissolved in a good solvent, a small amount of deionized water is slowly added dropwise to the solution under rapid stirring, the solution is stirred for a period of time, a large amount of deionized water is slowly added dropwise to fix the assembly, the solution is stirred for a period of time, and then the solution is transferred to a dialysis bag for dialysis. After dialysis, the micelle solution is diluted to a specific concentration to obtain a drug-loaded micelle solution. The drug-loaded micelle solution has excellent stability in an aqueous solution, can load a plurality of hydrophobic drugs, and can promote the release of the drug under acidic conditions.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a method for the self-assembly of amphiphilic polymers into drug-loaded micelles. Background Technology

[0002] Cancer is one of the most serious diseases threatening human life and health. According to the World Health Organization's (WHO) 2020 World Cancer Report, one in six deaths worldwide each year is due to cancer, with approximately 9.96 million deaths globally in 2020. The report also predicts that the number of cancer cases worldwide may increase further over the next two decades. Currently, traditional cancer treatments mainly consist of surgery, radiotherapy, and chemotherapy. Surgery is primarily for solid tumors and is difficult to treat metastatic malignant tumors. Chemotherapy can be applied to more types of tumors, but it lacks targeting and, while killing tumor cells, also has toxic effects on normal cells. Therefore, there is an urgent need for solutions that can reduce the toxic side effects of chemotherapy drugs without affecting their efficacy.

[0003] Nanoparticle drug delivery systems, leveraging their size advantage and the high-permeability, long-retention effect (EPR effect), can achieve drug accumulation at tumor sites while reducing their concentration in normal tissues. This effectively reduces the toxic side effects of chemotherapy drugs and has attracted widespread attention. However, most current nanoparticle drug delivery systems cannot selectively release drugs at tumor sites, leading to significant side effects.

[0004] In response to the above background, this invention proposes a polymeric micelle loaded with hydrophobic drugs, which is expected to achieve selective drug release in the slightly acidic environment of tumors. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing polymeric micelles loaded with hydrophobic drugs, which solves the problem of drug release in nanomedicine delivery systems and is expected to achieve selective drug release in the slightly acidic environment of tumors.

[0006] To achieve the above objectives, the present invention provides a method for preparing polymeric micelles loaded with hydrophobic drugs, comprising the following steps:

[0007] (1) Dissolve the amphiphilic polymer and the hydrophobic drug in a good solvent to obtain a good solvent solution;

[0008] (2) Under rapid stirring, deionized water is slowly added dropwise to a good solvent solution, or a good solvent solution is added dropwise to deionized water, so that the polymer self-assembles into drug-loaded micelles to obtain amphiphilic polymer drug-loaded micelles.

[0009] Preferably, the molar ratio of hydrophilic to hydrophobic segments of the amphiphilic polymer is 1:2-14.6:1, and more preferably 2.9:1-5.8:1.

[0010] Preferably, the hydrophilic segment of the amphiphilic polymer is the structure remaining after the tert-butyl protection of tert-butyl acrylate (tBA) is removed, and the hydrophobic segment is octadecyl acrylate (ODA).

[0011] Preferably, the mass ratio of the amphiphilic polymer to the hydrophobic drug is 20:2 to 20:10, and more preferably 20:2 to 20:4.

[0012] Preferably, the good solvent is acetonitrile or tetrahydrofuran.

[0013] Preferably, the ratio of amphiphilic polymer, good solvent and deionized water is 10mg-20mg: 1-2mL: 4-9mL.

[0014] Preferably, in step (2), under rapid stirring, a small amount of deionized water is slowly added dropwise to a good solvent solution, or a good solvent solution is added dropwise to a small amount of deionized water, and stirred for a period of time to promote polymer self-assembly. Then, a large amount of deionized water is slowly added dropwise to fix the assembly, and stirred for a period of time to obtain amphiphilic polymer drug-loaded micelles. The volume ratio of the small amount of deionized water to the large amount of deionized water is 0.2-0.5:4-8, for example, the small amount of deionized water is 200-500 μL, and the large amount of deionized water is 4-8 mL.

[0015] Preferably, the stirring speed is 1000-1200 rpm and the stirring time is 2-4 hours.

[0016] Preferably, the dropping rate is 1-5 mL / min, more preferably 1-2 mL / min.

[0017] Preferably, the self-assembled drug-loaded micelle solution is dialyzed in deionized water for 12-72 hours, more preferably 24-48 hours, with a dialyzed Mw of 3500-5000 Da.

[0018] Preferably, the water is changed every 4-24 hours during dialysis, more preferably every 6-8 hours.

[0019] Preferably, after dialysis, the micelle solution is diluted to a specific concentration (0.5-1 mg / mL) to obtain a drug-loaded micelle solution.

[0020] Preferably, castor oil is also added, and the amphiphilic polymer, the hydrophobic drug and the castor oil are co-dissolved in a good solvent, wherein the mass ratio of castor oil to hydrophobic drug is 1:3-10:3, preferably 2:3-5:3.

[0021] Preferably, the hydrophobic drug is docetaxel, doxorubicin, camptothecin, nilosin, prednisolone acetate, or vitamin A palmitate.

[0022] Specifically, the amphiphilic polymer and the hydrophobic drug are dissolved in a good solvent, and deionized water (200-500 μL) is added dropwise at 1-5 mL / min, preferably 1-2 mL / min, at 1000-1200 rpm. The mixture is stirred for 1-2 h to promote polymer self-assembly. Then, a large amount of water (4-8 mL) is slowly added dropwise while stirring for 1-2 h to fix the assemblies. The solution is then transferred to a dialysis bag (Mw = 3500-5000 Da) and dialyzed for 12-72 h, preferably 24-48 h. The water is changed every 4-24 h during dialysis, preferably 6-8 h. After dialysis, the micelle solution is diluted to 0.5-1 mg / mL to obtain the drug-loaded micelle solution.

[0023] Preferably, the method for preparing the amphiphilic polymer includes the following steps:

[0024] 1) In the presence of a catalyst, acetonitrile and 3-amino-1-propanol were used to synthesize the monomer 2-methyloxazine;

[0025] Preferably, in the presence of a catalyst, acetonitrile reacts with 3-amino-1-propanol at 80-115°C for 24-72 h, and the fraction at 120-130°C is collected by atmospheric distillation to obtain the monomer 2-methyloxazine.

[0026] Preferably, the catalyst is zinc acetate, 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.

[0027] Preferably, the molar ratio of acetonitrile to 3-amino-1-propanol is 1:0.9-1:1.1, and more preferably 1:1.1.

[0028] Preferably, the reaction temperature is 95-105℃ and the reaction time is 40-48h.

[0029] Specifically, acetonitrile and zinc acetate are added to a three-necked flask and heated to 80-115°C, preferably 95-105°C. After reflux of the acetonitrile, 3-amino-1-propanol is slowly added dropwise using a constant-pressure dropping funnel. The reaction is carried out for 24-72 hours, preferably 40-48 hours, to obtain crude 2-methyloxazine (MeOZI). The crude product is then subjected to atmospheric distillation, and the fraction collected at 120-130°C is used to obtain a colorless and transparent liquid, which is the purified 2-methyloxazine.

[0030] 2) The 2-methyloxazine monomer was subjected to cationic ring-opening polymerization to obtain polymethyloxazine;

[0031] Preferably, after dehydration treatment of the 2-methyloxazine (MeOZI) monomer and initiator, the reaction is carried out in a microwave reactor at 140-150℃ for 30-60 min to perform cationic ring-opening polymerization (CROP) to obtain polymethyloxazine (PMeOZI).

[0032] Preferably, the initiator is at least one of methyl p-toluenesulfonate (MeOTs), methyl trifluoromethanesulfonate, and acetyl chloride.

[0033] Preferably, the molar ratio of monomer MeOZI to initiator is 40:1-175:1, more preferably 88:1-100:1.

[0034] Preferably, the solvent for the reaction is acetonitrile.

[0035] Preferably, calcium hydride is used to remove water from the 2-methyloxazine monomer, and molecular sieves are used to remove water from the initiator.

[0036] Preferably, a 1-2M methanol solution of KOH is added at 0-5℃, with the molar ratio of KOH to initiator being 1:1-2:1, preferably 1:1. The polymerization is terminated by stirring at room temperature for 12-24 hours.

[0037] Specifically, firstly, the monomer MeOZI and the initiator undergo dehydration pretreatment. MeOZI dehydration: Calcium hydride is added to MeOZI, and the mixture is distilled at atmospheric pressure, collecting the fraction at 120-130℃; the amount of calcium hydride used is 5%-10% of the MeOZI mass. Initiator dehydration: The molecular sieve is activated in a muffle furnace at 400-500℃ for 1-3 hours. The activated molecular sieve is then placed in the initiator sample and treated at 30-40℃ for 24-48 hours before use. Cationic ring-opening polymerization of MeOZI: MeOZI and MeOTs are dissolved in acetonitrile and added to a Biotage microwave vial. The solution is reacted in a microwave reactor at 140℃ for 50 minutes, changing from colorless to yellow. Subsequently, a 1-2M KOH methanol solution is added at 0-5℃, and the mixture is stirred at room temperature for 12-24 hours to terminate the polymerization. The solvent was removed by rotary evaporation, the residual polymer was dissolved in chloroform, and purified by precipitation in cold n-hexane 3-5 times. The supernatant was removed by centrifugation at 3000-8000 rpm, and the polymer was dried in vacuum at 40℃ for 48 h to obtain polymethyloxazine (PMeOZI).

[0038] 3) PMeOZI was hydrolyzed under acidic conditions to obtain linear polypropyleneimine (lPPI).

[0039] Preferably, the hydrogen ion concentration before hydrolysis is 1M-10M, more preferably 3.4-4.4M; hydrochloric acid is used to provide acidic conditions.

[0040] Preferably, the hydrolysis temperature is 110-120℃ and the hydrolysis time is 6-72h, more preferably 18-36h.

[0041] Specifically, PMeOZI is dissolved in hot water at 80-100℃, heated to 110-120℃ and refluxed for 15-20 min, followed by the addition of concentrated hydrochloric acid. The reaction time is 6-72 h, preferably 18-36 h, and the hydrogen ion concentration before hydrolysis is 1M-10M, preferably 3.4-4.4M. The solvent is removed by rotary evaporation, and the residual solid is redissolved in hot water. NaOH solution is added to neutralize the solution to pH 9-10, resulting in the precipitation of a large amount of pale yellow precipitate. The precipitate is centrifuged at 3000-8000 rpm, the supernatant is discarded, and the process is repeated 3-5 times. The precipitate is then dried under vacuum at 40-65℃ for 24-48 h to obtain linear polypropyleneimine (1PPI).

[0042] 4) Graft tert-butyl acrylate (tBA) and octadecyl acrylate (ODA) onto the main chain of lPPI via Michael addition reaction to obtain an amphiphilic polymer precursor (PPI-tBA-ODA).

[0043] Preferably, the molar ratio of tBA to ODA is 1:2.0-14.6:1, and more preferably 2.9:1-5.8:1.

[0044] Preferably, the molar ratio of the secondary amine group of PPI to the sum of tBA and ODA is 1:0.9-1:1.1, and more preferably 1:1.

[0045] Preferably, the reaction temperature is 35-45℃ and the reaction time is 24-48h.

[0046] Preferably, the reaction solvent is a mixture of ethanol and tetrahydrofuran, with a volume ratio of ethanol to tetrahydrofuran of 1:1-10:1, preferably 2:1-5:1.

[0047] Preferably, PPI and tBA are dissolved in ethanol, and then a THF solution of ODA is added. The mixture is stirred at 35-45°C for 24-48 hours to obtain an amphiphilic polymer precursor (PPI-tBA-ODA).

[0048] Specifically, PPI and tBA are dissolved in ethanol, and then a THF solution of ODA is added. The mixture is stirred at 35-45°C for 24-48 hours, and the solution is rotary evaporated to obtain a concentrated solution. Precipitation is then performed 3-5 times in a suitable precipitant. The supernatant is removed by centrifugation at 3000-8000 rpm, and the solution is vacuum dried at 40°C for 48 hours. The precipitant is methanol, water, n-hexane, or diethyl ether, preferably methanol or water.

[0049] 5) The amphiphilic polymer precursor is hydrolyzed under acidic conditions to remove the tert-butyl group, yielding the amphiphilic polymer.

[0050] Preferably, PPI-tBA-ODA is dissolved in a mixed solution of acid and corresponding solvent to dissolve the polymer, stirred at 35-45°C for 2-6 hours, precipitated in diethyl ether, centrifuged to remove the supernatant, and dried to obtain deprotected PPI-tBA-ODA, which is the amphiphilic polymer.

[0051] Preferably, the acid is acetic acid, glycolic acid, or trifluoroacetic acid, and the solvent is n-hexane, chloroform, dichloromethane, tetrahydrofuran, or acetone, preferably n-hexane or chloroform.

[0052] Preferably, the volume ratio of acid to solvent in the mixed solution of acid and solvent is 1:1 to 1:2.

[0053] Specifically, PPI-tBA-ODA is dissolved in a mixed solution of acid and corresponding solvent to dissolve the polymer. The mixture is stirred at 35-45℃ for 2-6 hours, precipitated in diethyl ether 3-5 times, centrifuged at 3000-8000 rpm to remove the supernatant, and then vacuum dried at 30-40℃ for 24-48 hours to remove the protection.

[0054] The drug-loaded micelle solution prepared in this invention was subjected to performance tests, and the drug release behavior of the drug-loaded micelle solution was different in different buffer solutions.

[0055] First, the hydrophilic-hydrophobic ratio of the amphiphilic polymer was selected. Specifically, four different hydrophilic-hydrophobic ratios (1:2, 2.9:1, 5.8:1, and 14.6:1, respectively) of the amphiphilic polymer (20 mg) and the hydrophobic drug (4 mg) were dissolved in tetrahydrofuran (THF) (2 mL). Deionized water (400 μL) was slowly added dropwise under rapid stirring for 2 h. Then, a large amount of water (8 mL) was slowly added dropwise to fix the assembly, and the mixture was stirred for 2 h. The solution was then transferred to a dialysis bag and dialyzed for 24 h. After dialysis, the micelle solution was diluted to 1 mg / mL, and the particle size and zeta potential were tested. Figure 1 As shown. Simultaneously, the drug loading and encapsulation efficiency were calculated, such as... Figure 2 As shown.

[0056] Secondly, the ratio of polymer to drug was screened. Specifically, an amphiphilic polymer (20 mg) and a hydrophobic drug (0, 2, 3, 4, and 10 mg, respectively) were dissolved in 2 mL of THF. Under rapid stirring, 400 μL of deionized water was slowly added dropwise, and the mixture was stirred for 2 hours. Then, 8 mL of water was slowly added dropwise to fix the assembly, and the mixture was stirred for another 2 hours. The solution was then transferred to a dialysis bag and dialyzed for 24 hours. After dialysis, the micelle solution was diluted to 1 mg / mL, and the DLS and zeta potentials were measured. Figure 3 As shown, calculate the drug loading and encapsulation efficiency, as follows. Figure 4 As shown.

[0057] Next, the drug release curves of the drug-loaded micelles were tested. Specifically, 8 mL of a 1 mg / mL drug-loaded micelle solution was placed in a dialysis bag and then placed in 20 mL of PB buffer (pH 7.4) and acetate-sodium acetate buffer (pH 5.0), respectively. At different time intervals, 2 mL of the reaction solution was taken, diluted with 2 mL of acetonitrile, and left overnight until completely dissolved. Then, all the dialysis buffer was replaced, and the absorbance of the sample at 230 nm was measured. The absorbance was then substituted into the standard curve to calculate the concentration. The drug release curve is shown below. Figure 5 As shown, the drug release rate is faster under acidic conditions.

[0058] Next, the stability of the drug-loaded micelle solution was tested. Specifically, a 1 mg / mL drug-loaded micelle solution was stored at 4°C, and the particle size and PDI of the drug-loaded micelles were tested periodically. The results are as follows: Figure 6 As shown, drug-loaded micelles can maintain size stability within 14 days at 4°C.

[0059] Finally, the ability of drug-loaded micelles to load different hydrophobic drugs was tested. Specifically, an amphiphilic polymer (20 mg) and a hydrophobic drug (3 mg) were dissolved in 2 mL of a good solvent. 400 μL of deionized water was slowly added dropwise under rapid stirring for 2 h. Then, 8 mL of water was slowly added dropwise to immobilize the assembly, and the mixture was stirred for 2 h. The solution was then transferred to a dialysis bag and dialyzed for 24 h. After dialysis, the micelle solution was diluted to 1 mg / mL.

[0060] This invention prepares amphiphilic polymer-loaded drug micelles with hydrophilic zwitterions, improving the water solubility and stability of the carrier, while hydrophobic octadecyl groups are used to load hydrophobic drugs. After dissolving the polymer in a good solvent, water is slowly added. The hydrophobic segments aggregate internally to form a hydrophobic core, while the hydrophilic segments are exposed to form a hydrophilic shell. The self-assembled micelle structure can be used to load hydrophobic drugs, enhancing their solubility in aqueous solutions. Simultaneously, due to the large number of tertiary amines in the polymer structure, it exhibits pH responsiveness, accelerating micelle disintegration under acidic conditions and promoting drug release.

[0061] Beneficial effects:

[0062] 1) Due to the equal amount of positive and negative charges, the hydrophilic zwitterionic structure can bind more strongly to water molecules through ionic solvation, thus enhancing the hydrophilicity of the polymer. The self-assembled micelles have a hydrophilic zwitterionic shell and exhibit good stability in aqueous solutions.

[0063] 2) The hydrophobic octadecyl structure can support hydrophobic drugs. During self-assembly, the hydrophobic drug and the octadecyl structure are encapsulated in the core. This amphiphilic polymer can increase the solubility of hydrophobic drugs in aqueous solutions.

[0064] 3) The tertiary amino groups on the polymer backbone will be protonated under acidic conditions, which will promote the disintegration of micelles. Therefore, acidic conditions can promote the release of drugs from drug-loaded micelles. Attached Figure Description

[0065] Figure 1 : The 1H NMR spectrum of the monomer 2-methyloxazine.

[0066] Figure 2 : 1H NMR spectrum of polymethyloxazine (PMeOZI).

[0067] Figure 3 : 1H NMR spectrum of linear polypropyleneimine (lPPI).

[0068] Figure 4 : 1H NMR spectra of four amphiphilic polymer precursors (PPI-tBA-ODA) with hydrophilic-hydrophobic ratios, where (a)-(d) correspond to Examples 1-4 respectively.

[0069] Figure 5 : 1H NMR spectra of four amphiphilic polymers (PPICB-ODA) with hydrophilic-hydrophobic ratios, where (a)-(d) correspond to Examples 1-4 respectively.

[0070] Figure 6 Four ratios of blank and drug-loaded micelles: (a) particle size and (b) zeta potential.

[0071] Figure 7 (a) Encapsulation efficiency and (b) drug loading of four polymers with different proportions.

[0072] Figure 8 (a) Particle size and (b) zeta potential of different polymers with DTX ratios.

[0073] Figure 9 (a) Encapsulation efficiency and (b) drug loading of different polymers with DTX ratios.

[0074] Figure 10 Drug release curves at different pH values.

[0075] Figure 11 Stability of drug-loaded micelles at 4°C.

[0076] Figure 12 The effect of castor oil on the size and potential of drug-loaded micelles.

[0077] Figure 13 Particle size of Example 1 and Example 16.

[0078] Figure 14 Dorcetathione standard curve. Detailed Implementation

[0079] This invention provides a method for preparing drug-loaded micelles from amphiphilic polymers. The method includes the following steps: dissolving an amphiphilic polymer and a hydrophobic drug in a good solvent; slowly adding a small amount of deionized water, or adding a good solvent solution to a small amount of deionized water, under rapid stirring; stirring for a period of time; then slowly adding a large amount of deionized water to fix the assembly; stirring for a period of time; and finally transferring the solution to a dialysis bag for dialysis. After dialysis, the micelle solution is diluted to a specific concentration to obtain a drug-loaded micelle solution.

[0080] The following specific embodiments are for further explanation of the content of the present invention and should not be construed as limiting the present invention in any way.

[0081] Example 1

[0082] The preparation method of amphiphilic polymers with a hydrophilic-hydrophobic molar ratio (1:2) is shown below:

[0083] 1) Ultra-dry 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 80 °C. After reflux with acetonitrile, 3-amino-1-propanol (657 mmol, 50 mL) was slowly added dropwise using a constant-pressure dropping funnel. The reaction was allowed to proceed for 40 h to obtain 2-methyloxazine (MeOZI). The crude product was distilled at atmospheric pressure, and the fraction collected at 120–130 °C yielded a colorless, transparent liquid, MeOZI, in 35% yield. Figure 1 The structure of the monomer MeOZI was determined.

[0084] 2) First, the monomer MeOZI and the initiator methyl p-toluenesulfonate (MeOTs) were pretreated to remove water. MeOZI dehydration: 5% (w / w) calcium hydride was added to MeOZI, and the mixture was distilled at atmospheric pressure, collecting the fraction at 120-130℃. Initiator methyl p-toluenesulfonate (MeOTs) dehydration: 3A molecular sieves were activated in a muffle furnace at 400℃ for 3 hours. The activated molecular sieves were then placed in a 50mL beaker containing 15g of sample, ensuring the sieves covered the bottom of the beaker. The beaker was then treated at 30℃ for 24 hours before use. Cationic ring-opening polymerization of MeOZI (degree of polymerization 100): MeOZI (50mmol, 4.7mL), MeOTs (0.5mmol, 75.4μL), and ultra-dry acetonitrile (7.7mL) were mixed in a 20mL Biotage microwave vial. The solution was reacted in a microwave reactor at 140℃ for 50 minutes, changing from colorless to yellow. Subsequently, a 1M KOH methanol solution (0.25 mL) was added at 0 °C, and the polymerization was terminated by stirring overnight at room temperature. The solvent was removed by rotary evaporation, and the residual polymer was dissolved in chloroform (5 mL), precipitated and purified three times in cold n-hexane (45 mL), and dried under vacuum at 40 °C for 48 h to obtain a pale yellow viscous solid with a yield of 95%. Figure 2 The structure of PMiOZI was determined.

[0085] 3) PMeOZI (22.67 mmol, 2.2469 g) was dissolved in 17 mL of hot water at 100 °C. The solution was heated to 120 °C and refluxed for 15 min. Then, 30 mL of 37% hydrochloric acid was added, and the reaction was allowed to proceed for 36 h. The solvent was removed by rotary evaporation, and the remaining solid was redissolved in 77 mL of hot water. 0.1 M NaOH solution was added to neutralize the solution to pH 9, resulting in a large amount of pale yellow precipitate. The precipitate was centrifuged at 3000 rpm, the supernatant was discarded, and this process was repeated three times. The precipitate was then dried under vacuum at 65 °C for 48 h to obtain orange-yellow crystalline solid polypropyleneimine (PPI) with a yield of 98%. Figure 3 The structure of lPPI has been determined.

[0086] 4) Dissolve PPI (300 mg, 5.25 mmol) and tBA (249 μL) in ethanol (15 mL), then add THF solution of ODA (1136 mg) (5 mL), mix at 35 °C for 48 h, rotary evaporate to obtain a concentrated solution (5 mL), precipitate three times in methanol (45 mL), centrifuge to remove the supernatant, and vacuum dry at 40 °C for 48 h. Figure 4 (a) The PPI-tBA-ODA structure was determined.

[0087] 5) Dissolve PPI-tBA-ODA (252 mg) in a mixed solution (4 mL) of TFA / n-hexane (1:1) to dissolve the polymer. Stir at 35°C for 6 h, precipitate three times in diethyl ether, centrifuge to remove the supernatant, and vacuum dry at 40°C for 48 h. Figure 5 (a) The PPICB-ODA structure was determined.

[0088] The preparation method of drug-loaded micelles is as follows:

[0089] An amphiphilic polymer (20 mg) and docetaxel (4 mg) in a hydrophilic-hydrophobic molar ratio (1:2) were dissolved in 2 mL of tetrahydrofuran (THF). Under stirring at 1000 rpm, 400 μL of deionized water was added dropwise at a rate of 1 mL / min for 2 h. Then, 8 mL of deionized water was added dropwise at a rate of 1 mL / min to fix the assembly, and the mixture was stirred for another 2 h. The solution was then transferred to a dialysis bag (Mw = 3500 Da), and the water was changed every 6 h for dialysis over 24 h. After dialysis, the micelle solution was diluted to 1 mg / mL. The particle size potential is as follows: Figure 6 As shown in Figure 1, the particle size is 256 nm, the potential is 82 mV, and the drug loading and encapsulation efficiency are as follows. Figure 7 As shown, the drug loading was 6% and the encapsulation efficiency was 28%. The preparation process of the drug-loaded micelles used a blank micelle prepared without docetaxel as a control, denoted as Blank 1.

[0090] Example 2

[0091] The difference between this embodiment and Example 1 is that the amounts of tert-butyl acrylate and octadecyl acrylate are different, and the hydrophilic-hydrophobic molar ratio of the amphiphilic polymer is 2.9:1 (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 structure of PPICB-ODA was determined. Particle size potentials are as follows: Figure 6 As shown in Figure 2, the particle size is 604 nm, the potential is 48 mV, and the drug loading and encapsulation efficiency are as follows. Figure 7 As shown, the drug loading is 15% and the encapsulation efficiency is 73%.

[0092] Example 3

[0093] The difference between this embodiment and Example 1 is that the amounts of tert-butyl acrylate and octadecyl acrylate are different, the precipitant is different, the hydrophilic-hydrophobic molar ratio of the amphiphilic polymer is 5.8:1 (638 μL of tert-butyl acrylate and 249 mg of octadecyl acrylate), and the precipitant is water. Figure 4 (c) The PPI-tBA-ODA structure was determined. Figure 5 (c) The structure of PPICB-ODA was determined. Particle size potentials are as follows: Figure 6 As shown in Figure 3, the drug loading is 474 nm in diameter and 44 mV in potential. The drug loading and encapsulation efficiency are as follows: Figure 7 As shown, the drug loading was 14% and the encapsulation efficiency was 68%. The preparation process of the drug-loaded micelles used a blank micelle prepared without docetaxel as a control, denoted as blank 3.

[0094] Example 4

[0095] The difference between this embodiment and Example 1 is that the amounts of tert-butyl acrylate and octadecyl acrylate are different, the precipitant is different, the hydrophilic-hydrophobic molar ratio of the amphiphilic polymer is 14.6:1 (700 μL of tert-butyl acrylate and 109 mg of octadecyl acrylate), and the precipitant is water. Figure 4 (d) The PPI-tBA-ODA structure was determined. Figure 5 (d) The structure of PPICB-ODA was determined. Particle size potentials are as follows: Figure 6 As shown in Figure 1, the particle size is 219 nm, the potential is 21 mV, and the drug loading and encapsulation efficiency are as follows. Figure 7 As shown, the drug loading was 16% and the encapsulation efficiency was 79%. The preparation process of the drug-loaded micelles used a blank micelle prepared without docetaxel as a control, designated as blank 4.

[0096] Example 5

[0097] The amphiphilic polymer (20 mg) with a hydrophilic-hydrophobic molar ratio of 2.9:1 prepared in Example 2 and docetaxel (0 mg) were dissolved in 2 mL of tetrahydrofuran (THF). Under stirring at 1000 rpm, 400 μL of deionized water was added dropwise at a rate of 1 mL / min, and the mixture was stirred for 2 h. Then, 8 mL of deionized water was added dropwise at a rate of 1 mL / min to fix the assembly, and the mixture was stirred for 2 h. The solution was then transferred to a dialysis bag (Mw = 3500 Da), and the water was changed every 6 h for dialysis for 24 h. After dialysis, the micelle solution was diluted to 1 mg / mL. The particle size potential is as follows... Figure 8 As shown, the particle size is 150 nm and the potential is 38 mV.

[0098] Example 6

[0099] The difference between this embodiment and Embodiment 5 is that the mass of docetaxel is 2 mg. Particle size potential is as follows: Figure 8 As shown, the particle size is 254 nm, the potential is 76 mV, and the drug loading and encapsulation efficiency are as follows: Figure 9 As shown, the drug loading is 5% and the encapsulation efficiency is 53%.

[0100] Example 7

[0101] The difference between this embodiment and Example 5 is that the mass of docetaxel is 3 mg. Particle size potential is as follows: Figure 8 As shown, the particle size is 287 nm, the potential is 66 mV, and the drug loading and encapsulation efficiency are as follows: Figure 9 As shown, the drug loading is 7% and the encapsulation efficiency is 46%.

[0102] Example 8

[0103] The difference between this embodiment and Embodiment 5 is that the mass of docetaxel is 4 mg. Particle size potential is as follows: Figure 8 As shown, the particle size is 604 nm, the potential is 48 mV, and the drug loading and encapsulation efficiency are as follows. Figure 9 As shown, the drug loading is 15% and the encapsulation efficiency is 73%.

[0104] Example 9

[0105] The difference between this embodiment and Example 5 is that the mass of docetaxel is 10 mg. Particle size potential is as follows: Figure 8 As shown, the particle size is 1175 nm, the potential is 55 mV, and the drug loading and encapsulation efficiency are as follows: Figure 9 As shown, the drug loading is 20% and the encapsulation efficiency is 41%.

[0106] Example 10

[0107] The difference between this embodiment and Example 2 is that the good solvent is changed from tetrahydrofuran to acetonitrile.

[0108] Example 11

[0109] The amphiphilic polymer (20 mg) with a hydrophilic-hydrophobic molar ratio of 2.9:1 prepared in Example 2, along with docetaxel (4 mg) and castor oil (5 mg), was dissolved in tetrahydrofuran (THF) (2 mL). Under stirring at 1000 rpm, deionized water (400 μL) was added dropwise at a rate of 1 mL / min, and the mixture was stirred for 2 h. Then, deionized water (8 mL) was added dropwise at a rate of 1 mL / min to fix the assembly, and the mixture was stirred for 2 h. The solution was then transferred to a dialysis bag (Mw = 3500 Da), and the water was changed every 6 h for dialysis for 24 h. After dialysis, the micelle solution was diluted to 1 mg / mL. The particle size potential is as follows: Figure 12 As shown, the particle size is 260 nm and the potential is 65 mV. The addition of castor oil has no significant effect on the particle size and potential of the drug-loaded micelles.

[0110] Example 12

[0111] The amphiphilic polymer (20 mg) with a hydrophilic-hydrophobic molar ratio of 2.9:1 prepared in Example 2 was dissolved in tetrahydrofuran (THF) (2 mL) along with Nile Red (4 mg) and castor oil (5 mg). Under stirring at 1000 rpm, deionized water (400 μL) was added dropwise at a rate of 1 mL / min, and the mixture was stirred for 2 h. Then, deionized water (8 mL) was added dropwise at a rate of 1 mL / min to fix the assembly, and the mixture was stirred for 2 h. The solution was then transferred to a dialysis bag (Mw = 3500 Da), and the water was changed every 6 h for dialysis for 24 h. After dialysis, the micelle solution was diluted to 1 mg / mL.

[0112] Example 13

[0113] The difference between this embodiment and Embodiment 12 is that the encapsulated drug is doxorubicin.

[0114] Example 14

[0115] The difference between this embodiment and Embodiment 12 is that the encapsulated drug is vitamin A palmitate.

[0116] Example 15

[0117] The difference between this embodiment and Embodiment 12 is that the encapsulated drug is prednisolone acetate.

[0118] Example 16

[0119] The amphiphilic polymer (20 mg) with a hydrophilic-hydrophobic molar ratio (2.9:1) prepared in Example 2 was dissolved in tetrahydrofuran (THF) (2 mL) to obtain a tetrahydrofuran solution. Under stirring at 1000 rpm, the tetrahydrofuran solution was added dropwise to deionized water (400 μL) at a rate of 1 mL / min for 2 h. Then, deionized water (8 mL) was added dropwise at a rate of 1 mL / min to fix the assembly, and the mixture was stirred for 2 h. The solution was then transferred to a dialysis bag (Mw = 3500 Da), and the water was changed every 6 h for dialysis for 24 h. After dialysis, the micelle solution was diluted to 1 mg / mL. Particle size is as follows. Figure 13 As shown, the particle size is 3652 nm. Therefore, the water-drop-oil method is more suitable for the preparation of micelles in this invention than the oil-drop-water method.

[0120] Example 17

[0121] The drug release curves of the drug-loaded micelles were tested. 8 mL of the 1 mg / mL drug-loaded micelle solution prepared in Example 1 was placed in a dialysis bag (Mw = 3500 Da) and then placed in 20 mL of PB buffer (pH 7.4) and acetate-sodium acetate buffer (pH 5.0), respectively. At different time points (2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, and 48 h), 2 mL of the buffer outside the dialysis bag was taken, diluted with 2 mL of acetonitrile, and incubated overnight until completely dissolved. The absorbance of the sample at 230 nm was measured. After each sampling, the remaining buffer outside the dialysis bag was replaced with fresh 20 mL of PB buffer (pH 7.4) and acetate-sodium acetate buffer (pH 5.0). The absorbance of the sample at 230 nm was then substituted into... Figure 14 The standard curve is used to calculate its concentration. The release curve is as follows: Figure 10 As shown, the slightly acidic conditions at pH 5.0, compared to pH 7.4, allow for the release of more drugs, thus enabling the carrier to be selectively released within the tumor microenvironment.

[0122] Example 18

[0123] The stability of the drug-loaded micelle solution was tested. A 1 mg / mL solution of the drug-loaded micelle solution prepared in Example 1 was stored at 4°C, and the particle size and PDI of the drug-loaded micelles were tested periodically. The results are as follows: Figure 11 As shown, within 14 days, the drug-loaded micelles maintained dimensional stability at 4℃, with PDI all below 0.3 and uniform distribution.

Claims

1. A method for preparing polymeric drug-loaded micelles loaded with hydrophobic drugs, characterized in that, The method includes the following steps: (1) Dissolve the amphiphilic polymer and the hydrophobic drug in a good solvent to obtain a good solvent solution; (2) Under stirring conditions, add deionized water dropwise to a good solvent solution, or add a good solvent solution dropwise to deionized water, so that the polymer self-assembles into drug-loaded micelles; The molar ratio of hydrophilic to hydrophobic segments in the amphiphilic polymer is 1:2-14.6:1; the hydrophilic segment of the amphiphilic polymer is the structure remaining after the tert-butyl group is removed from tert-butyl acrylate, and the hydrophobic segment is octadecyl acrylate. The method for preparing the amphiphilic polymer includes the following steps: 1) In the presence of a catalyst, acetonitrile and 3-amino-1-propanol were used to synthesize the monomer 2-methyloxazine; 2) The 2-methyloxazine monomer was subjected to cationic ring-opening polymerization to obtain polymethyloxazine; 3) Polymethyloxazine is hydrolyzed under acidic conditions to yield linear polypropyleneimine; 4) Grafting tert-butyl acrylate and octadecyl acrylate onto the polypropylene imine backbone yields an amphiphilic polymer precursor; 5) The amphiphilic polymer precursor is hydrolyzed under acidic conditions to obtain the amphiphilic polymer.

2. The preparation method according to claim 1, characterized in that: The mass ratio of the amphiphilic polymer to the hydrophobic drug is 20:2 to 20:

10.

3. The preparation method according to claim 1, characterized in that: The good solvent is acetonitrile or tetrahydrofuran; the ratio of amphiphilic polymer, good solvent and deionized water is 10mg-20mg: 1-2mL: 4-9mL.

4. The preparation method according to claim 1, characterized in that: Castor oil is also added, and the amphiphilic polymer and hydrophobic drug are co-soluble with castor oil in a good solvent. The mass ratio of castor oil to hydrophobic drug is 1:3-10:

3.

5. The hydrophobic drug prepared according to claim 1 is docetaxel, doxorubicin, camptothecin, nilosin, prednisolone acetate, or vitamin A palmitate.

6. The preparation method according to claim 1, characterized in that: In step (2), under stirring conditions, a small amount of deionized water is first added to the good solvent solution, or a good solvent solution is added to a small amount of deionized water, and stirred, and then a large amount of deionized water is added and stirred; wherein, the volume ratio of the small amount of deionized water to the large amount of deionized water is 0.2-0.5:4-8.

7. The preparation method according to claim 1, characterized in that: The stirring speed was 1000-1200 rpm for 2-4 h; the dropping rate was 1-5 mL / min; the self-assembled drug-loaded micelle solution was dialyzed in deionized water for 12-72 h, and the molecular weight cutoff for dialyzing was 3500-5000 Da.

8. The preparation method according to claim 1, characterized in that: In step 1), the reaction temperature is 80-115℃, the reaction time is 24-72h, and the fraction collected at 120-130℃ is collected by atmospheric 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, zinc chloride, or zinc acetate dihydrate, and the molar ratio of acetonitrile to catalyst is 1:0.01-1:0.05; In step 2), after dehydrating the 2-methyloxazine monomer and initiator, the reaction is carried out in a microwave reactor at 140-150℃ for 30-60 min to perform cationic ring-opening polymerization to obtain polymethyloxazine; the initiator is at least one of methyl p-toluenesulfonate, methyl trifluoromethanesulfonate, and acetyl chloride, and the molar ratio of 2-methyloxazine monomer to initiator is 40:1-175:1; the reaction solvent is acetonitrile; calcium hydride is used to dehydrate the 2-methyloxazine monomer, and molecular sieves are used to dehydrate the initiator; In step 3), the hydrogen ion concentration before hydrolysis is 1-10 M, the reaction temperature is 110-120℃, and the reaction time is 6-72 h. In step 4), the molar ratio of tert-butyl acrylate to octadecyl acrylate is 1:2.0-14.6:1, and the molar ratio of the secondary amino group of polypropyleneimide to the sum of tert-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, methanol, diethyl ether, or n-hexane. In step 5), the amphiphilic polymer precursor is dissolved in a mixed solution of acid and solvent, stirred at 35-45°C for 2-6 hours, precipitated in diethyl ether, centrifuged to remove the supernatant, and dried to obtain the amphiphilic polymer; wherein the acid is acetic acid, glycolic acid, or trifluoroacetic acid, the solvent is n-hexane, chloroform, or dichloromethane, and the volume ratio of acid to solvent in the mixed solution of acid and solvent is 1:1-1:2.

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