Hydrophobic drug-loaded pH-responsive nano-micelle and preparation method thereof
By grafting carboxyl groups and forming hydrazone bonds into nanomicelles, the drug loading rate and stability of hydrophobic drugs were improved, enabling specific drug release in tumor regions.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHENGDU HENGMEISHENG BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-01-19
- Publication Date
- 2026-04-21
AI Technical Summary
Existing nanomicelles loaded with hydrophobic drugs have low drug loading rates and are difficult to accumulate in tumor regions.
A pH-responsive nanomicelle loaded with a hydrophobic drug was prepared by grafting carboxyl groups onto a methoxy polyethylene glycol-polycaprolactone amphiphilic block copolymer to form an N-hydroxysuccinimide ester group, followed by a nucleophilic substitution reaction with a compound containing a hydrazine group, and then a condensation reaction with a hydrophobic drug containing a ketone group to form a hydrazone bond.
It improves drug loading rate and drug loading stability, making the drug easier to release under acidic conditions and able to specifically accumulate in the tumor area.
Smart Images
Figure CN121891301A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the fields of polymer biomaterials and drug delivery technology, and more specifically, to a pH-responsive nanomicelle loaded with a hydrophobic drug and its preparation method. Background Technology
[0002] PEG-PCL is an amphiphilic block copolymer composed of hydrophilic methoxy polyethylene glycol (mPEG) and hydrophobic polycaprolactone (PCL) linked by chemical bonds. Utilizing its ability to self-assemble into core-shell structured nanomicelles in aqueous solution, it can be used as a carrier for water-transported drugs. Specifically, the hydrophobic PCL core can load hydrophobic drugs (such as paclitaxel (PTX) and doxorubicin, used to treat tumors), while the hydrophilic mPEG shell provides steric stability and long-term cycling properties. However, existing nanomicelles loaded with hydrophobic drugs suffer from low drug loading rates, and their passive long-term cycling properties make them difficult to specifically accumulate in tumor regions (which are typically acidic). Summary of the Invention
[0003] The purpose of this application is to provide a pH-responsive nanomicelle loaded with hydrophobic drugs and a method for preparing the same. The pH-responsive nanomicelles loaded with hydrophobic drugs prepared by this method have a high drug loading rate.
[0004] The embodiments of this application are implemented as follows: In a first aspect, embodiments of this application provide a method for preparing pH-responsive nanomicelles loaded with hydrophobic drugs, comprising the following steps: S1. Acidification reaction is carried out by dissolving a methoxy polyethylene glycol-polycaprolactone amphiphilic block copolymer, an acid anhydride, and a catalyst in anhydrous dichloromethane, wherein the molecular weight of the methoxy polyethylene glycol is 2000 Da~3000 Da and the molecular weight of the polycaprolactone is 8000 Da~16000 Da, to obtain an amphiphilic block copolymer containing carboxyl groups; S2. The amphiphilic block copolymer containing carboxyl groups is activated to form N-hydroxysuccinimide ester groups; S3. The amphiphilic block copolymer containing N-hydroxysuccinimide ester groups undergoes a nucleophilic substitution reaction with a compound containing a hydrazine group to obtain an amphiphilic block copolymer containing an acylhydrazine group; S4. The amphiphilic block copolymer containing an acylhydrazine group undergoes a condensation reaction with a hydrophobic drug containing a ketone group to obtain a nanomicelle precursor; S5. The nanomicelle precursor was dialyzed in water to remove impurities and allow it to self-assemble into micelles, thus obtaining pH-responsive nanomicelles loaded with hydrophobic drugs.
[0005] In the above technical solution, during the acidification reaction, anhydrous dichloromethane is used as the anhydrous organic solvent, and the molecular weights of methoxy polyethylene glycol and polycaprolactone are simultaneously limited to the aforementioned ranges. This allows for the grafting of more carboxyl groups during the acidification reaction (the more carboxyl groups in the early stage, the more hydrazide groups in the later stage, and the more hydrophobic drugs that can be chemically bonded during the condensation reaction with hydrophobic drugs containing ketone groups). This results in micelles with a high drug loading rate. At the same time, most of the drugs are chemically grafted onto the hydrophobic core of polycaprolactone, which also gives the micelles high drug loading stability (specifically, the drugs are easily released under acidic conditions rather than being difficult to release under non-acidic conditions). This allows most of the pH-responsive nanomicelles carrying hydrophobic drugs to reach the tumor area before being released.
[0006] In some alternative embodiments, the molecular weight of methoxy polyethylene glycol is 2000 Da to 2500 Da, and the molecular weight of polycaprolactone is 8000 Da to 12000 Da.
[0007] In the above technical solution, the molecular weights of methoxy polyethylene glycol and polycaprolactone are limited to the above range, so that the two have a more suitable molecular weight ratio, thereby making the pH-responsive nanomicelles loaded with hydrophobic drugs have a higher drug loading rate.
[0008] In some alternative embodiments, step S1 includes: S11 dissolving the amphiphilic block copolymer in anhydrous dichloromethane to obtain an amphiphilic block copolymer solution; S12 dissolving the acid anhydride and catalyst in the amphiphilic block copolymer solution and performing an acidification reaction under stirring conditions to obtain a first mixed solution; S13 adding the first mixed solution to a precipitant for settling and solid-liquid separation to obtain an amphiphilic block copolymer precipitate containing carboxyl groups.
[0009] In the above technical solution, the stepwise dissolution and mixing method in the above order during the early stage of the acidification reaction can improve the grafting rate of carboxyl groups.
[0010] In some alternative embodiments, the anhydride is selected from succinic anhydride, and the molar ratio of succinic anhydride to amphiphilic block copolymer is (1.5~2):1; or / and the catalyst is selected from 4-dimethylaminopyridine, and the molar ratio of catalyst to amphiphilic block copolymer is (0.2~0.5):1.
[0011] In the above technical solution, the molar ratio of succinic anhydride to amphiphilic block copolymer is limited to the above range, that is, the appropriate excess of succinic anhydride can maximize the grafting rate of carboxyl groups without wasting raw materials; the molar ratio of catalyst to amphiphilic block copolymer is limited to the above range, that is, providing a more appropriate amount of catalyst also helps to improve the grafting rate of carboxyl groups.
[0012] In some alternative embodiments, the molar ratio of the amphiphilic block copolymer to the volume of anhydrous dichloromethane is 1 mmol : (20 mL to 30 mL); or / and, the precipitant is selected from diethyl ether, and the volume ratio of the precipitant to anhydrous dichloromethane is (100 to 150) : (20 to 30).
[0013] In the above technical solution, the molar ratio of the amphiphilic block copolymer to the volume of anhydrous dichloromethane is limited to the above range so that the two have a more suitable dosage ratio. The suitable dosage ratio not only enables the amphiphilic block copolymer to dissolve quickly and completely, but also enables the formed amphiphilic block copolymer solution to have a more suitable concentration, which facilitates the subsequent efficient reaction of grafting carboxyl groups. Limiting the volume ratio of the precipitant to anhydrous dichloromethane to the above range helps the formed amphiphilic block copolymer containing carboxyl groups to precipitate quickly and completely.
[0014] In some alternative embodiments, step S2 includes: dissolving the amphiphilic block copolymer containing carboxyl groups, the condensing agent, and the activator in a first anhydrous organic solvent to obtain a second mixed solution; stirring the second mixed solution in an ice-water bath for 0.5 h to 1 h, and then stirring it in a 20°C to 30°C for 1.5 h to 3 h, so that the carboxyl groups form N-hydroxysuccinimide ester groups.
[0015] In the above technical solution, during the activation treatment step, the second mixed solution is first stirred and reacted under ice-water bath conditions for the above-mentioned time, and then stirred and reacted at 20℃~30℃ for the above-mentioned time. This can effectively reduce the generation of by-products, so that the carboxyl group can be more completely formed into N-hydroxysuccinimide ester group, so that more hydrophobic drugs can be grafted on in the future, thereby improving the drug loading rate.
[0016] In some alternative embodiments, the molar ratio of the carboxyl-containing amphiphilic block copolymer to the condensing agent is 1:(1.2~1.5); or / and, the molar ratio of the carboxyl-containing amphiphilic block copolymer to the activator is 1:(1.0~1.2).
[0017] In the above technical solution, the ratio of the molar amount of the amphiphilic block copolymer containing carboxyl groups to the molar amount of the condensing agent, and the ratio of the molar amount of the amphiphilic block copolymer containing carboxyl groups to the molar amount of the activator are respectively limited within the above range, so that the carboxyl groups can be more thoroughly formed into N-hydroxysuccinimide ester groups, so that more hydrophobic drugs can be grafted on them in the future, thereby improving the drug loading rate.
[0018] In some alternative embodiments, step S3 includes: adding an amphiphilic block copolymer having an N-hydroxysuccinimide ester group, a hydrazine-containing compound, and a pH adjuster to a second anhydrous organic solvent to obtain a third mixed solution, wherein the pH of the third mixed solution is 7.1 to 8.5; and then subjecting the third mixed solution to a nucleophilic substitution reaction under stirring conditions at 20°C to 30°C to obtain an amphiphilic block copolymer containing an acylhydrazine group.
[0019] In the above technical solution, the third mixed solution contains a pH adjuster and limits the pH value of the third mixed solution to the above range (i.e., weakly alkaline), which can effectively neutralize the acidic N-hydroxysuccinimide formed during the reaction (an acidic environment will inhibit the nucleophilicity of acyl hydrazide), thereby increasing the grafting rate of acyl hydrazide group and thus increasing the drug loading rate.
[0020] In some optional embodiments, step S4 includes: S41 dissolving the amphiphilic block copolymer containing an acylhydrazine group in a third anhydrous organic solvent to obtain a fourth mixed solution; and dissolving the hydrophobic drug containing a ketone group in a buffer solution with a pH of 4.5 to 5.5 to obtain a hydrophobic drug solution; S42 mixing the fourth mixed solution and the hydrophobic drug solution and adjusting the pH to 4.5 to 5.5 to obtain a fifth mixed solution; and subjecting the fifth mixed solution to a condensation reaction at 20°C to 30°C under light-protected and stirred conditions to obtain a nanomicelle precursor.
[0021] In the above technical solution, the hydrophobic drug containing ketone groups is dissolved in a buffer solution with a pH of 4.5-5.5. The weakly acidic environment can reduce the probability of degradation of the hydrophobic drug containing ketone groups, thereby improving the chemical stability of the drug. At the same time, the weakly acidic conditions are also conducive to the subsequent condensation reaction between the hydrophobic drug and the hydrazide group on the amphiphilic block copolymer, thereby improving the drug grafting rate. Mixing the fourth mixed solution and the hydrophobic drug solution and adjusting the pH to 4.5-5.5 can reduce the probability of degradation of the hydrophobic drug containing ketone groups. At the same time, this pH range is conducive to the subsequent condensation reaction between the hydrophobic drug and the hydrazide group on the amphiphilic block copolymer, thereby improving the drug grafting rate.
[0022] Secondly, embodiments of this application provide a pH-responsive nanomicelle loaded with a hydrophobic drug, prepared using the preparation method provided in the first aspect embodiment.
[0023] In the above technical solution, the pH-responsive nanomicelles loaded with hydrophobic drugs are prepared by the preparation method provided in the first aspect embodiment, so as to give them a high drug loading rate. Attached Figure Description
[0024] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.
[0025] Figure 1 A process flow diagram of a method for preparing pH-responsive nanomicelles loaded with hydrophobic drugs, provided for embodiments of this application; Figure 2 This is an electron microscope image of the nanomicelles in Example 1 of this application; Figure 3 This is a particle size distribution diagram of the nanomicelles in Example 1 of this application; Figure 4 This is a physical image of the nanomicelles in Example 1 of this application. Detailed Implementation
[0026] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.
[0027] It should be noted that the terms "and / or" in this application, such as "feature 1 and / or feature 2", all refer to the three cases of "feature 1" alone, "feature 2" alone, and "feature 1" plus "feature 2".
[0028] In addition, in the description of this application, unless otherwise stated, "one or more" means two or more; the range of "numerical value a to numerical value b" includes the two endpoints "a" and "b"; and "unit of measurement" in "numerical value a to numerical value b + unit of measurement" represents the "unit of measurement" of both "numerical value a" and "numerical value b".
[0029] In existing technologies, when using methoxy polyethylene glycol-polycaprolactone amphiphilic block copolymers as carriers to load hydrophobic drugs, they are usually simply mixed with the hydrophobic drugs. That is, the coating of hydrophobic drugs is only achieved through the self-assembly of spheres, resulting in low loading rate and weak binding. Furthermore, it does not have pH responsiveness, so most of the drugs are released during passive circulation and are difficult to accumulate in the tumor area.
[0030] In this application, the loading of hydrophobic drugs is mainly achieved through chemical bonding. Specifically, carboxyl groups are first grafted onto polycaprolactone, and then the amphiphilic block copolymer containing carboxyl groups is activated to form N-hydroxysuccinimide ester groups. Then, the amphiphilic block copolymer containing N-hydroxysuccinimide ester groups undergoes a nucleophilic substitution reaction with a compound containing hydrazine groups to obtain an amphiphilic block copolymer containing hydrazide groups. Then, the amphiphilic block copolymer containing hydrazide groups undergoes a condensation reaction with a hydrophobic drug containing ketone groups to obtain a nanomicelle precursor (that is, most of the hydrophobic drug is loaded onto the polycaprolactone segments of the amphiphilic block copolymer through chemical bonding, specifically the hydrazone bond formed by the condensation reaction of hydrazide groups and ketone groups, which is a pH-sensitive bond that is easily broken under acidic conditions). Finally, the precursor is self-assembled into spheres by dialysis in water. This stage also encapsulates a small amount of hydrophobic drug (most existing technologies only have this stage).
[0031] The following is a detailed description of a pH-responsive nanomicelle loaded with a hydrophobic drug and its preparation method, according to an embodiment of this application.
[0032] In a first aspect, embodiments of this application provide a method for preparing pH-responsive nanomicelles loaded with hydrophobic drugs, comprising the following steps: S1. An acidification reaction is carried out by dissolving a methoxy polyethylene glycol-polycaprolactone amphiphilic block copolymer, an acid anhydride, and a catalyst in anhydrous dichloromethane, wherein the molecular weight of the methoxy polyethylene glycol is 2000 Da to 3000 Da (e.g., but not limited to any one of 2000 Da, 2200 Da, 2400 Da, 2600 Da, 2800 Da, and 3000 Da, or any range between the two), and the molecular weight of the polycaprolactone is 8000 Da to 16000 Da (e.g., but not limited to any one of 8000 Da, 10000 Da, 12000 Da, 14000 Da, 15000 Da, and 16000 Da, or any range between the two), to obtain an amphiphilic block copolymer containing carboxyl groups; S2. The amphiphilic block copolymer containing carboxyl groups is activated to form N-hydroxysuccinimide ester groups; S3. An amphiphilic block copolymer containing an N-hydroxysuccinimide ester group is subjected to a nucleophilic substitution reaction with a compound containing a hydrazine group to obtain an amphiphilic block copolymer containing an acylhydrazine group; S4 The amphiphilic block copolymer containing an acylhydrazine group is subjected to a condensation reaction with a hydrophobic drug containing a ketone group to obtain a nanomicelle precursor; S5 The nanomicelle precursor is dialyzed in water to remove impurities and allow the nanomicelle precursor to form micelles through self-assembly, thereby obtaining pH-responsive nanomicelles loaded with a hydrophobic drug.
[0033] In this application, anhydrous dichloromethane is used as the anhydrous organic solvent, and the molecular weights of methoxy polyethylene glycol and polycaprolactone are simultaneously limited to the aforementioned ranges. This allows for the grafting of more carboxyl groups during the acidification reaction (the more carboxyl groups in the early stage, the more hydrazide groups in the later stage, and the more hydrophobic drugs that can be chemically bonded during the condensation reaction with hydrophobic drugs containing ketone groups). This results in micelles with a high drug loading rate. At the same time, most of the drug is grafted onto the hydrophobic core of polycaprolactone through chemical bonds (specifically, hydrazone bonds formed by the condensation reaction of hydrazide groups and ketone groups, which are pH-sensitive bonds and easily broken under acidic conditions). This also results in micelles with high drug loading stability (specifically, drugs are easily released under acidic conditions but not easily released under non-acidic conditions). This allows most of the pH-responsive nanomicelles carrying hydrophobic drugs to reach the tumor area before being released.
[0034] It should be noted that drug loading stability refers to the degree of firmness of the binding between the drug and the micelles. The higher the drug loading stability, the less likely the drug in the micelles is to be released or leaked out during storage, which helps to prevent its failure. At the same time, it can also reduce the amount of drug released during circulation and before reaching the lesion area, thereby increasing its accumulation in the lesion area.
[0035] As an example, methoxy polyethylene glycol has a molecular weight of 2000 Da to 2500 Da (e.g., but not limited to any one of 2000 Da, 2100 Da, 2200 Da, 2300 Da, 2400 Da and 2500 Da or any range between two), and polycaprolactone has a molecular weight of 8000 Da to 12000 Da (e.g., but not limited to any one of 8000 Da, 10000 Da and 12000 Da or any range between two).
[0036] In this embodiment, the molecular weights of methoxy polyethylene glycol and polycaprolactone are limited to the above-mentioned ranges to achieve a more suitable molecular weight ratio, thereby resulting in a higher drug loading rate for the pH-responsive nanomicelles loaded with hydrophobic drugs.
[0037] As an example, step S1 includes: S11 dissolving the amphiphilic block copolymer in anhydrous dichloromethane to obtain an amphiphilic block copolymer solution; S12 dissolving the acid anhydride and catalyst in the amphiphilic block copolymer solution and performing an acidification reaction under stirring conditions to obtain a first mixed solution; S13 adding the first mixed solution to a precipitant for static treatment and solid-liquid separation treatment to obtain an amphiphilic block copolymer precipitate containing carboxyl groups.
[0038] In this embodiment, the stepwise dissolution and mixing method in the above order during the early stage of the acidification reaction can improve the grafting rate of carboxyl groups.
[0039] In other possible implementations, the amphiphilic block copolymer, acid anhydride, and catalyst may be dissolved together in anhydrous dichloromethane.
[0040] It should be noted that there are no restrictions on the types of acid anhydrides and catalysts, and they can be selected and set in accordance with the conventional methods used in this field.
[0041] As an example, the anhydride is selected from succinic anhydride, and the molar ratio of succinic anhydride to the amphiphilic block copolymer is (1.5~2):1, for example, but not limited to any point value or range between any two of the ratios of 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1 and 2:1.
[0042] In this embodiment, the molar ratio of succinic anhydride to amphiphilic block copolymer is limited to the above range, that is, succinic anhydride is appropriately in excess, which can maximize the grafting rate of carboxyl groups without wasting raw materials.
[0043] As an example, the catalyst is selected from 4-dimethylaminopyridine, and the molar ratio of the catalyst to the amphiphilic block copolymer is (0.2~0.5):1, for example, but not limited to any one of the ratios of 0.2:1, 0.3:1, 0.4:1 and 0.5:1 or any range between the two.
[0044] In this embodiment, the molar ratio of the catalyst to the amphiphilic block copolymer is limited to the above range, which provides a more suitable amount of catalyst and also helps to improve the grafting rate of carboxyl groups.
[0045] As an example, the molar amount of the amphiphilic block copolymer is any one of the following points or any range between the two: 1 mmol: 20 mL, 1 mmol: 22 mL, 1 mmol: 24 mL, 1 mmol: 26 mL, 1 mmol: 28 mL and 1 mmol: 30 mL of anhydrous dichloromethane.
[0046] In this embodiment, the molar ratio of the amphiphilic block copolymer to the volume of anhydrous dichloromethane is limited to the above-mentioned range so that the two have a more suitable dosage ratio. The suitable dosage ratio not only enables the amphiphilic block copolymer to dissolve quickly and completely, but also enables the formed amphiphilic block copolymer solution to have a more suitable concentration, which facilitates the subsequent efficient reaction of grafting carboxyl groups.
[0047] It should be noted that there are no restrictions on the type of precipitant, and it can be selected and set in accordance with the conventional methods used in this field.
[0048] As an example, the precipitant is selected from diethyl ether, and the volume ratio of the precipitant to anhydrous dichloromethane is (100~150):(20~30), for example, but not limited to any one of the ratios of 100:20, 100:30, 150:20 and 150:30, or any range between the two.
[0049] In this embodiment, limiting the volume ratio of the precipitant to anhydrous dichloromethane within the above-mentioned range helps the formed amphiphilic block copolymer containing carboxyl groups to precipitate quickly and completely.
[0050] It should be noted that for acidification processes that are not specifically described or limited, they can be carried out in accordance with conventional processes in this field.
[0051] As an example, the acidification reaction is carried out under closed and inert protection conditions, wherein the acidification reaction time is 12 h to 24 h, the stirring speed is 300 rpm to 500 rpm, and the reaction temperature is 25℃ to 50℃.
[0052] As an example, after solid-liquid separation, the process also includes washing the precipitate multiple times with diethyl ether to remove residual acid anhydride and catalyst.
[0053] As an example, step S2 includes: dissolving the carboxyl-containing amphiphilic block copolymer, condensing agent, and activator in a first anhydrous organic solvent to obtain a second mixed solution; stirring the second mixed solution in an ice-water bath for 0.5 h to 1 h (e.g., but not limited to any one of 0.5 h, 0.6 h, 0.7 h, 0.8 h, 0.9 h, and 1 h, or any range between any two), and then stirring the solution in a 20°C to 30°C for 1.5 h to 3 h (e.g., but not limited to any one of 1.5 h, 1.8 h, 2 h, 2.5 h, and 3 h, or any range between any two) to form N-hydroxysuccinimide ester groups from the carboxyl groups.
[0054] In this embodiment, during the activation process, the second mixed solution is first stirred and reacted in an ice-water bath for the aforementioned duration, and then stirred and reacted again at 20°C to 30°C for the aforementioned duration. This effectively reduces the formation of byproducts, allowing the carboxyl groups to more thoroughly form N-hydroxysuccinimide ester groups, so that more hydrophobic drugs can be grafted onto them subsequently, thereby improving the drug loading rate.
[0055] As an example, the molar ratio of the amphiphilic block copolymer containing carboxyl groups to the molar ratio of the condensing agent is 1:(1.2~1.5), for example, but not limited to any one of the ratios of 1:1.2, 1:1.3, 1:1.4 and 1:1.5 or any range between the two.
[0056] In this embodiment, the molar ratio of the amphiphilic block copolymer containing carboxyl groups to the molar ratio of the condensing agent is limited to the above range so that the carboxyl groups can be more thoroughly formed into N-hydroxysuccinimide ester groups, so that more hydrophobic drugs can be grafted on subsequently, thereby improving the drug loading rate.
[0057] As an example, the molar ratio of the amphiphilic block copolymer containing carboxyl groups to the molar ratio of the activator is 1:(1.0~1.2), for example, but not limited to any one of the ratios of 1:1.0, 1:1.05, 1:1.1, 1:1.15 and 1:1.2, or any range between the two.
[0058] In this embodiment, the molar ratio of the amphiphilic block copolymer containing carboxyl groups to the molar ratio of the activator is limited to the above range so that the carboxyl groups can be more thoroughly formed into N-hydroxysuccinimide ester groups, so that more hydrophobic drugs can be grafted on subsequently, thereby improving the drug loading rate.
[0059] It should be noted that since the molecular weight of amphiphilic block copolymers containing carboxyl groups is mainly provided by the skeleton of the amphiphilic block copolymer, when calculating the molar amount of amphiphilic block copolymers containing carboxyl groups, the amphiphilic block copolymers without carboxyl groups should be used as the standard.
[0060] It should be noted that for any steps in the activation process that are not specifically described or limited, they can be performed in accordance with conventional processes in this field.
[0061] As an example, the condensing agent is selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, denoted as EDC-HCl; the activator is selected from N-hydroxysuccinimide, denoted as NHS; and the first anhydrous organic solvent is selected from at least one of DCM, THF and DMF.
[0062] As an example, the activation process is carried out under closed and inert protection conditions, wherein the stirring speed is 300 rpm to 500 rpm.
[0063] As an example, after the stirring reaction is completed, the process also includes filtering with an organic membrane of 0.45 μm and drying the filtrate. Filtration removes insoluble impurities, and drying yields a dry powder, which facilitates the accurate weighing of raw materials in subsequent steps.
[0064] As an example, step S3 includes: adding an amphiphilic block copolymer having an N-hydroxysuccinimide ester group, a compound containing an acylhydrazine group, and a pH adjuster to a second anhydrous organic solvent to obtain a third mixed solution, wherein the pH of the third mixed solution is 7.1 to 8.5 (e.g., but not limited to any one of pH values or any range between 7.1, 7.2, 7.5, 7.8, 8.0, 8.2, and 8.5); and then subjecting the third mixed solution to a nucleophilic substitution reaction under stirring conditions at 20°C to 30°C to obtain an amphiphilic block copolymer containing an acylhydrazine group.
[0065] In this embodiment, the third mixed solution contains a pH adjuster and the pH value of the third mixed solution is limited to the above-mentioned range (i.e., weakly alkaline), which can effectively neutralize the acidic N-hydroxysuccinimide formed during the reaction (an acidic environment inhibits the nucleophilicity of acylhydrazide), thereby increasing the grafting rate of the acylhydrazide group and thus increasing the drug loading rate.
[0066] It should be noted that the types of compounds containing hydrazine groups, the second anhydrous organic solvent, and the pH adjuster are not limited and can be selected and set in accordance with conventional practices in the field.
[0067] As an example, the compound containing a hydrazine group is selected as adipic acid dihydrazide, denoted as ADH, and the molar ratio of the amphiphilic block copolymer having an N-hydroxysuccinimide ester group to adipic acid dihydrazide is 1:(1.2~1.5), for example, but not limited to any one of the ratios of 1:1.2, 1:1.3, 1:1.4 and 1:1.5 or any range between the two.
[0068] As an example, the second anhydrous organic solvent is selected from DMF, and the molar amount of the amphiphilic block copolymer having N-hydroxysuccinimide ester groups is 1 mmol:(20 mL to 30 mL) of DMF, for example, but not limited to, any one of 1 mmol:20 mL, 1 mmol:22 mL, 1 mmol:24 mL, 1 mmol:26 mL, 1 mmol:28 mL and 1 mmol:30 mL or any range between the two.
[0069] As an example, the pH adjuster is selected from triethylamine, and the molar ratio of the amphiphilic block copolymer having an N-hydroxysuccinimide ester group to the pH adjuster is 1:(0.5~1), for example, but not limited to any point value or range between any two of the ratios of 1:0.5, 1:0.6, 1:0.8 and 1:1.
[0070] It should be noted that since the molecular weight of amphiphilic block copolymers with N-hydroxysuccinimide ester groups is mainly provided by the amphiphilic block copolymer backbone, when calculating the molar amount of amphiphilic block copolymers containing carboxyl groups, the amphiphilic block copolymers without N-hydroxysuccinimide ester groups shall be used as the standard.
[0071] As an example, after the nucleophilic substitution reaction is completed, the process includes precipitation, solid-liquid separation, washing, and drying. The precipitation process includes adding the reaction solution to anhydrous diethyl ether at a volume ratio of 1:1 and allowing it to stand. The washing process includes washing the collected precipitate with anhydrous diethyl ether. The drying process includes drying the washed solid in a vacuum drying oven at 30°C for 12 h to obtain a dried powder of the amphiphilic block copolymer with N-hydroxysuccinimide ester groups.
[0072] As an example, step S4 includes: S41 dissolving the amphiphilic block copolymer containing an acylhydrazine group in a third anhydrous organic solvent to obtain a fourth mixed solution; and dissolving the hydrophobic drug containing a ketone group in a buffer solution with a pH of 4.5 to 5.5 (e.g., but not limited to any one of pH values of 4.5, 4.8, 5.0, 5.2, and 5.5, or any range between two), to obtain a hydrophobic drug solution; S42 mixing the fourth mixed solution and the hydrophobic drug solution and adjusting the pH to 4.5 to 5.5 (e.g., but not limited to any one of pH values of 4.5, 4.8, 5.0, 5.2, and 5.5, or any range between two), to obtain a fifth mixed solution; and subjecting the fifth mixed solution to a condensation reaction at 20°C to 30°C under light-protected and stirred conditions to obtain a nanomicelle precursor.
[0073] In this embodiment, the hydrophobic drug containing a ketone group is dissolved in a buffer solution with a pH of 4.5-5.5. The weakly acidic environment can reduce the probability of degradation of the hydrophobic drug containing a ketone group, thereby improving the chemical stability of the drug. At the same time, the weakly acidic conditions are also conducive to the subsequent condensation reaction between the hydrophobic drug and the hydrazide group on the amphiphilic block copolymer, thereby improving the drug grafting rate. Mixing the fourth mixed solution and the hydrophobic drug solution and adjusting the pH to 4.5-5.5 can reduce the probability of degradation of the hydrophobic drug containing a ketone group. At the same time, this pH range is conducive to the subsequent condensation reaction between the hydrophobic drug and the hydrazide group on the amphiphilic block copolymer, thereby improving the drug grafting rate.
[0074] As an example, the condensation reaction was carried out in a light-proof, sealed, and inert gas-protected environment for 12 to 24 hours, with a stirring speed of 200 to 300 rpm.
[0075] It should be noted that there are no restrictions on the types of hydrophobic drugs, third anhydrous organic solvents, and buffer solutions; they can be selected and set according to conventional practices in the field.
[0076] As an example, the hydrophobic drug is selected from doxorubicin hydrochloride, and the molar ratio of the amphiphilic block copolymer containing an acylhydrazine group to the hydrophobic drug is 1:(1.2~1.5), for example, but not limited to any point value or range between any two of the ratios of 1:1.2, 1:1.3, 1:1.4 and 1:1.5.
[0077] As an example, the third anhydrous organic solvent is selected from anhydrous DMSO, and the molar amount of the amphiphilic block copolymer containing the hydrazide group is 1 mmol: (10 mL to 15 mL) of DMSO, for example, but not limited to, any one of 1 mmol: 10 mL, 1 mmol: 11 mL, 1 mmol: 12 mL, 1 mmol: 13 mL, 1 mmol: 14 mL and 1 mmol: 15 mL or any range between the two.
[0078] As an example, the buffer is selected from 0.1 M acetate buffer, and the volume of the amphiphilic block copolymer containing the hydrazide group and the acetate buffer is 1 mmol: (10 mL to 15 mL), for example, but not limited to, any one of 1 mmol: 10 mL, 1 mmol: 11 mL, 1 mmol: 12 mL, 1 mmol: 13 mL, 1 mmol: 14 mL and 1 mmol: 15 mL or any range between the two.
[0079] It should be noted that since the molecular weight of amphiphilic block copolymers containing hydrazide groups is mainly provided by the skeleton of the amphiphilic block copolymer, when calculating the molar amount of amphiphilic block copolymers containing carboxyl groups, the amphiphilic block copolymers without hydrazide groups should be used as the standard.
[0080] As an example, step S5 includes: transferring the solution containing the nanomicelle precursor in S4 to a dialysis bag with a molecular weight cutoff of 3500 Da and then to a container containing deionized water for dialysis for 24 h, with the deionized water being replaced every 6 h, in order to remove free hydrophobic drugs and small molecule impurities and to allow the nanomicelle precursor to form micelles through self-assembly, thereby obtaining pH-responsive nanomicelles loaded with hydrophobic drugs.
[0081] It should be noted that, unless otherwise specified or limited, the processes or steps in the preparation of pH-responsive nanomicelles loaded with hydrophobic drugs can be carried out in accordance with conventional processes in this field.
[0082] As an example, a process flow diagram for the preparation of pH-responsive nanomicelles loaded with hydrophobic drugs is exemplarily provided. Figure 1 .
[0083] Secondly, embodiments of this application provide a pH-responsive nanomicelle loaded with a hydrophobic drug, prepared using the preparation method provided in the first aspect embodiment.
[0084] In this application, the pH-responsive nanomicelles loaded with hydrophobic drugs are prepared using the preparation method provided in the first aspect embodiment to achieve a high drug loading rate.
[0085] The features and performance of this application will be further described in detail below with reference to the embodiments.
[0086] Example 1 This application provides a method for preparing pH-responsive nanomicelles loaded with hydrophobic drugs, comprising the following steps: S1 contains 1 mmol of an amphiphilic block copolymer (specifically mPEG). 2000 -PCL 10000 Dissolve succinic anhydride and 4-dimethylaminopyridine in 20 mL of anhydrous dichloromethane after drying with molecular sieves to obtain an amphiphilic block copolymer solution; then dissolve 2 mmol of succinic anhydride and 0.4 mmol of 4-dimethylaminopyridine in the amphiphilic block copolymer solution and stir at 400 rpm, room temperature (25℃), sealed and under nitrogen atmosphere for 24 h to obtain a first mixed solution; add the first mixed solution to 100 mL of diethyl ether and let stand for 30 min. After standing, filter using a Buchner funnel and collect the precipitate; wash the collected precipitate three times with diethyl ether to remove residual succinic anhydride and 4-dimethylaminopyridine, and then dry the precipitate in a vacuum drying oven at 30℃ for 12 h to obtain a carboxyl-containing amphiphilic block copolymer powder (i.e., mPEG). 2000 -PCL 10000 -COOH).
[0087] S2 will add 1 mmol of mPEG 2000 -PCL 10000 -COOH was dissolved in 20 mL of anhydrous dichloromethane after dehydration by molecular sieve, and then 1.5 mmol of EDC-HCl and 1.2 mmol of NHS were added to obtain a second mixed solution. The second mixed solution was stirred in an ice-water bath for 1 h under a sealed nitrogen atmosphere, and then stirred at 25 °C for 2 h to allow the carboxyl groups to form N-hydroxysuccinimide ester groups. The resulting reaction solution was then filtered through a 0.45 μm organic filter membrane to remove insoluble impurities. The filtrate was then concentrated to dryness under reduced pressure, and the powder was sealed and stored at -20 °C under anhydrous conditions to obtain a dry powder of amphiphilic block copolymer containing N-hydroxysuccinimide ester groups (i.e., mPEG). 2000 -PCL 10000 -COO-NHS).
[0088] S3 will add 1 mmol of mPEG 2000 -PCL 10000-COO-NHS was dissolved in 20 mL of anhydrous DMF after dehydration by molecular sieve to obtain a third mixed solution. Then, 1.5 mmol of adipic acid dihydrazide and triethylamine were added to the third mixed solution to adjust the pH to 7.5. The mixture was then stirred at 400 rpm, room temperature (25°C), sealed, and under a nitrogen atmosphere for 3 h. The reaction solution was then added to 100 mL of diethyl ether and allowed to stand for 30 min. After standing, the mixture was filtered using a Buchner funnel, and the precipitate was collected. The collected precipitate was washed three times with diethyl ether to remove residual adipic acid dihydrazide and NHS byproducts. The precipitate was then dried in a vacuum drying oven at 30°C for 12 h to obtain a dry powder of an amphiphilic block copolymer containing hydrazide groups (i.e., mPEG). 2000 -PCL 10000 -CONH-NH2).
[0089] S4 will contain 1 mmol of mPEG 2000 -PCL 10000 -CONH-NH2 was dissolved in 10 mL of anhydrous DMSO after being dehydrated by molecular sieves to obtain a fourth mixed solution; simultaneously, 1.5 mmol of doxorubicin hydrochloric acid was dissolved in an acetate buffer solution with a pH of 5.0 to obtain a hydrophobic drug solution; the fourth mixed solution and the hydrophobic drug solution were mixed and the pH was adjusted to 5.0 to obtain a fifth mixed solution; the fifth mixed solution was subjected to a condensation reaction at 25 °C under light-protected, sealed, and stirred conditions (250 rpm) for 18 h to obtain the nanomicelle precursor.
[0090] S5 The solution containing the nanomicelle precursor was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and then to a container containing deionized water for dialysis for 24 h. The deionized water was replaced every 6 h to remove free hydrophobic drugs and small molecule impurities and to allow the nanomicelle precursor to form micelles through self-assembly, thus obtaining pH-responsive nanomicelles loaded with hydrophobic drugs.
[0091] The preparation processes of the subsequent embodiments and comparative examples are all carried out in accordance with Example 1. In order to better understand the differences between the embodiments and comparative examples, some parameters are summarized and explained in the form of a table, see Table 1 for details.
[0092] Table 1
[0093] Test case (1) Morphology and particle size test Test method: The nanomicelles prepared in Example 1 were used as samples, and morphological images of the samples were acquired using electron microscopy. At the same time, the particle size of the samples was measured using a particle size analyzer.
[0094] See Figure 2 and Figure 3 It can be seen that the nanomicelles are uniformly dispersed particles, and the particle size results show that the average particle size of the micelles is around 100 nm.
[0095] (2) Collection of physical images Test method: The nanomicelles prepared in Example 1 were used as samples and then transferred to a transparent glass bottle for photographing.
[0096] See Figure 4 It can be seen that the nanomicelles present as a uniform white liquid.
[0097] (3) Drug loading rate test Test method: Nanomicelles were prepared according to the preparation methods of Examples 1-11 and Comparative Examples 1-4, respectively. Then, all the external liquid from the dialysis bag in step S5 was collected and used as the test sample. The total mass of free doxorubicin in the external liquid (i.e., the mass of doxorubicin not loaded into the nanomicelles) was then determined by liquid chromatography. The specific test conditions were as follows: Column: C18 reversed-phase column (e.g., Agilent ZORBAX SB-C18, 4.6×250 mm); Mobile phase: methanol-water-trifluoroacetic acid (55:45:0.1, v / v / v); Flow rate: 1.0 mL / min; Column temperature: 30℃; Detection wavelength: 480 nm; Standard curve preparation: 0.1~10 μg / mL doxorubicin standard solution was prepared, and 20 μL was injected to plot the peak area-concentration curve; The calculation method was the same as that of the external standard method. Then, according to the formula: drug loading rate (%) = (additional drug amount - free drug amount) / additional drug amount × 100%, the test results are finally summarized in Table 2.
[0098] Table 2
[0099] Referring to Tables 1 and 2, when anhydrous dichloromethane is used as the organic solvent and the molecular weights of mPEG and PCL are simultaneously limited to the ranges of 2000 Da~3000 Da and 8000 Da~16000 Da, respectively, the pH-responsive nanomicelles loaded with hydrophobic drugs prepared by the former exhibit higher drug loading rates compared to other molecular weight combinations. Specifically, when the molecular weight of mPEG is in the range of 2000 Da~2500 Da, and the molecular weight of PCL is simultaneously limited to the range of 8000 Da~12000 Da, the pH-responsive nanomicelles loaded with hydrophobic drugs prepared achieve even higher drug loading rates.
[0100] (4) In vitro release performance test of the sample Test method: The nanomicelles prepared in Example 1 were used as samples. The cumulative release rate of samples of the same mass at different time points in systems with pH values of 7.4, 6.5 and 6.0 was then tested. Specifically, the dialysis bag method was used for testing. The specific method can be referred to as the drug loading rate test. The difference is that the cumulative drug release rate (%) = cumulative drug release amount / drug loading amount × 100%, where the drug loading amount refers to the total amount of drug loaded by the micelles in the initial stage. Finally, the test results are summarized in Table 3.
[0101]
[0102] As shown in Table 3, the cumulative release rate of the nanomicelles at physiological pH (7.4) for 72 h was only 22.3%, indicating that they can remain stable during passive circulation, allowing most of the drug to reach the tumor area and accumulate through circulation. Then, the hydrazone bonds (-CONHNH2) in the nanomicelles break under acidic conditions, allowing the drug accumulated in the tumor area to be rapidly released and act on the lesion area.
[0103] The embodiments described above are some, but not all, of the embodiments of this application. The detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.
Claims
1. A method for preparing pH-responsive nanomicelles loaded with hydrophobic drugs, characterized in that, Includes the following steps: S1. Acidification reaction is carried out by dissolving methoxy polyethylene glycol-polycaprolactone amphiphilic block copolymer, acid anhydride and catalyst in anhydrous dichloromethane, wherein the molecular weight of methoxy polyethylene glycol is 2000 Da~3000 Da and the molecular weight of polycaprolactone is 8000 Da~16000 Da, so as to obtain an amphiphilic block copolymer containing carboxyl groups. S2 activates the amphiphilic block copolymer containing carboxyl groups so that the carboxyl groups form N-hydroxysuccinimide ester groups; S3 involves a nucleophilic substitution reaction between an amphiphilic block copolymer containing an N-hydroxysuccinimide ester group and a compound containing a hydrazine group to obtain an amphiphilic block copolymer containing an acylhydrazine group. S4 The amphiphilic block copolymer containing hydrazide groups is condensed with a hydrophobic drug containing ketone groups to obtain a nanomicelle precursor. S5 The nanomicelle precursor is dialyzed in water to remove impurities and allow the nanomicelle precursor to self-assemble into micelles, thereby obtaining pH-responsive nanomicelles loaded with hydrophobic drugs.
2. The method for preparing pH-responsive nanomicelles loaded with hydrophobic drugs according to claim 1, characterized in that, The methoxy polyethylene glycol has a molecular weight of 2000 Da to 2500 Da, and the polycaprolactone has a molecular weight of 8000 Da to 12000 Da.
3. The method for preparing pH-responsive nanomicelles loaded with hydrophobic drugs according to claim 1, characterized in that, Step S1 includes: S11 Dissolve the amphiphilic block copolymer in anhydrous dichloromethane to obtain an amphiphilic block copolymer solution; S12 Dissolve the acid anhydride and catalyst in the amphiphilic block copolymer solution and carry out an acidification reaction under stirring conditions to obtain a first mixed solution; S13 The first mixed solution is added to a precipitant for static treatment and solid-liquid separation treatment to obtain an amphiphilic block copolymer precipitate containing carboxyl groups.
4. The method for preparing pH-responsive nanomicelles loaded with hydrophobic drugs according to claim 3, characterized in that, The acid anhydride is selected from succinic anhydride, and the molar ratio of the succinic anhydride to the amphiphilic block copolymer is (1.5~2):1; Or / and, the catalyst is selected from 4-dimethylaminopyridine, and the molar ratio of the catalyst to the amphiphilic block copolymer is (0.2~0.5):
1.
5. The method for preparing pH-responsive nanomicelles loaded with hydrophobic drugs according to claim 4, characterized in that, The molar ratio of the amphiphilic block copolymer to the volume of the anhydrous dichloromethane is 1 mmol: (20 mL~30 mL). Or / and, the precipitant is selected from diethyl ether, and the volume ratio of the precipitant to the anhydrous dichloromethane is (100~150):(20~30).
6. The method for preparing pH-responsive nanomicelles loaded with hydrophobic drugs according to any one of claims 1 to 5, characterized in that, Step S2 includes: The amphiphilic block copolymer containing carboxyl groups, the condensing agent, and the activator are dissolved in a first anhydrous organic solvent to obtain a second mixed solution. The second mixed solution is first stirred and reacted in an ice-water bath for 0.5 h to 1 h, and then stirred and reacted at 20℃ to 30℃ for 1.5 h to 3 h, so that the carboxyl groups form N-hydroxysuccinimide ester groups.
7. The method for preparing pH-responsive nanomicelles loaded with hydrophobic drugs according to claim 6, characterized in that, The molar ratio of the amphiphilic block copolymer containing carboxyl groups to the molar ratio of the condensing agent is 1:(1.2~1.5). Or / and, the molar ratio of the amphiphilic block copolymer containing carboxyl groups to the molar ratio of the activator is 1:(1.0~1.2).
8. The method for preparing pH-responsive nanomicelles loaded with hydrophobic drugs according to any one of claims 1 to 5, characterized in that, Step S3 includes: An amphiphilic block copolymer containing N-hydroxysuccinimide ester, a compound containing hydrazine, and a pH adjuster are added to a second anhydrous organic solvent to obtain a third mixed solution, wherein the pH of the third mixed solution is 7.1 to 8.5; then the third mixed solution is subjected to a nucleophilic substitution reaction under stirring conditions at 20°C to 30°C to obtain an amphiphilic block copolymer containing hydrazine.
9. The method for preparing pH-responsive nanomicelles loaded with hydrophobic drugs according to any one of claims 1 to 5, characterized in that, Step S4 includes: S41 Dissolve the amphiphilic block copolymer containing hydrazide groups in a third anhydrous organic solvent to obtain a fourth mixed solution; and dissolve the hydrophobic drug containing ketone groups in a buffer solution with a pH of 4.5 to 5.5 to obtain a hydrophobic drug solution; S42 The fourth mixed solution and the hydrophobic drug solution are mixed and the pH value is adjusted to 4.5~5.5 to obtain the fifth mixed solution; the fifth mixed solution is subjected to a condensation reaction at 20℃~30℃ under light-proof and stirring conditions to obtain the nanomicelle precursor.
10. A pH-responsive nanomicelle loaded with a hydrophobic drug, characterized in that, It is prepared by any one of the preparation methods described in claims 1 to 9.