Single molecular weight precision polyoxyethylene castor oil derivatives, methods of making, nanoparticles, and uses

Abstract

This application provides a single-molecular-weight precisely polyoxyethylene castor oil derivative, its preparation method, nanoparticles, and applications, belonging to the field of materials technology. This single-molecular-weight precisely polyoxyethylene castor oil derivative has the structural formula shown in formula (I) or formula (II): Formula (I), Formula (II), where x=y=z=4~100, w=4~100, a=1~10, b=0~5, c=1~10, d=0~5, e=1~10, f=0~5, g=0~5, h=1~10. This derivative possesses both water solubility and drug-load solubilizing ability, enabling the formation of uniformly sized and stable nanoparticles, improving the solubility of poorly soluble drugs. Simultaneously, its precise molecular structure effectively reduces the risks of hemolysis, allergies, and cytotoxicity caused by free surfactants, exhibiting higher biocompatibility during injection administration.

Description

Technical Field

[0001] This application relates to the field of materials technology, and in particular to a single molecular weight polyoxyethylene castor oil derivative, its preparation method, nanoparticles, and applications. Background Technology

[0002] Currently, commercially available formulations use polyoxyethylene castor oil (Cremophor EL) and ethanol in a 1:1 volume ratio, then dissolve antitumor drugs in this mixture at specific concentrations. Cremophor EL is a nonionic surfactant with typical amphiphilic characteristics; its polyethylene glycol portion is hydrophilic, while its three fatty acid tails are hydrophobic, forming micelles in water. The polyethylene glycol portion forms the hydrophilic shell, while the fatty acid tails encapsulate antitumor drugs such as paclitaxel in the middle, thus solubilizing the antitumor drugs. However, commercially available Cremophor EL is produced by polymerizing castor oil and ethylene oxide. It is actually a complex mixture with many different degrees of polymerization of polyethylene glycol, different types and amounts of fatty acid substitutions, and oxidation products, and there are also differences between different batches of the product. The lack of a defined structure increases its biological uncertainty in vivo, thereby increasing clinical risks. Summary of the Invention

[0003] In view of this, in order to at least partially solve at least one of the aforementioned technical problems, this application provides a single molecular weight precision polyoxyethylene castor oil derivative, a preparation method, nanoparticles, and applications.

[0004] According to one embodiment of this application, a single molecular weight precision polyoxyethylene castor oil derivative is provided, having a structural formula as shown in formula (I) or formula (II):

[0005] Formula (I),

[0006] Formula (II), where x=y=z=4~100, w=4~100, a=1~10, b=0~5, c=1~10, d=0~5, e=1~10, f=0~5, g=0~5, h=1~10, and R is selected from any one of hydroxyl, methoxy, amino, carboxyl, and maleimide.

[0007] According to another embodiment of this application, a method for preparing a single molecular weight precision polyoxyethylene castor oil derivative is provided, wherein the method for preparing the compound shown in formula (I) includes:

[0008] The compound shown in formula (A) with a single molecular weight of precisely polyethylene glycol was subjected to a nucleophilic substitution reaction with the compound shown in formula (B), and after removing the protecting group, the compound shown in formula (C) was obtained.

[0009]

[0010] Formula (A) Formula (B)

[0011] The compound shown in formula (C) and the compound shown in formula (D) are subjected to esterification to obtain the compound shown in formula (E), where PG represents a protecting group;

[0012]

[0013] Equation (C) Equation (D)

[0014] The compound shown in formula (E) was deprotected to obtain the compound shown in formula (I).

[0015]

[0016] Equation (E)

[0017] According to another embodiment of this application, a method for preparing a single molecular weight precision polyoxyethylene castor oil derivative is provided, wherein the method for preparing the compound shown in formula (II) includes:

[0018] The single molecular weight polyethylene glycol structure shown in formula (F) is esterified with the compound shown in formula (G) to obtain the compound shown in formula (H), where PG represents a protecting group;

[0019]

[0020] Equation (F) Equation (G)

[0021] The compound shown in formula (H) is deprotected to obtain the compound shown in formula (II).

[0022]

[0023] Formula (H)

[0024] According to another embodiment of this application, a nanoparticle is provided, which is assembled from a single molecular weight polyoxyethylene castor oil derivative by means of microfluidics, nano-flash delivery, mechanical dispersion, thin film hydration or ultrasonic dispersion.

[0025] According to another aspect of this application, an application of nanoparticles in antitumor drug delivery is provided.

[0026] According to the embodiments of this application, by precisely controlling the length of polyoxyethylene chain segments and the branched structure, the molecular weight and molecular configuration are uniformly controllable, overcoming the problems of large product quality fluctuations and difficulty in reproducing key physicochemical properties caused by the wide molecular weight distribution of commercial polyoxyethylene castor oil. This derivative has both excellent water solubility and drug loading and solubilizing ability, and can form nanoparticles with uniform and stable particle size, thereby improving the solubility of poorly soluble drugs. At the same time, its precise molecular structure effectively reduces the risks of hemolysis, allergy and cytotoxicity caused by free surfactants, and exhibits higher biosafety when administered by injection. Attached Figure Description

[0027] The above and other objects, features and advantages of this application will become clearer from the following description of embodiments with reference to the accompanying drawings, in which:

[0028] Figure 1 The mass spectrum of the compound of formula 3 prepared in Example 1 of this application;

[0029] Figure 2 The mass spectrum of the compound of formula 4 prepared in Example 1 of this application;

[0030] Figure 3 The mass spectrum of the compound of Formula 9 prepared in Example 1 of this application;

[0031] Figure 4 The 1H NMR spectrum of the compound of Formula 9 prepared in Example 1 of this application;

[0032] Figure 5 The mass spectrum of commercial polyoxyethylene castor oil, Comparative Example 1 of this application;

[0033] Figure 6 The gel permeation chromatograms are of polyoxyethylene castor oil in Comparative Example 1 and Example 1 of this application;

[0034] Figure 7 This is a particle size distribution diagram of the single molecular weight polyoxyethylene castor oil derivative nanoparticles prepared in Example 2 of this application.

[0035] Figure 8 This is a particle size distribution diagram of the commercial polyoxyethylene castor oil nanoparticles prepared in Comparative Example 2 of this application.

[0036] Figure 9 The results are shown in Figure 1 for the particle size and polydispersity index of the different nanoparticles used in Application Example 1 of this application.

[0037] Figure 10 The graph shows the cytotoxicity results of different nanoparticles used in Application Example 1 of this application against 4T1 tumor cells;

[0038] Figure 11 The graph shows the cytotoxicity results of different nanoparticles used in Application Example 1 of this application on normal 3T3 cells;

[0039] Figure 12 The graph shows the results of in vitro paclitaxel (PTX) release assays using different nanoparticles in Application Example 2 of this application. Detailed Implementation

[0040] The embodiments of this application will now be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of this application. In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the embodiments of this application for ease of explanation. However, it will be apparent that one or more embodiments may be implemented without these specific details. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concepts of this application.

[0041] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. The term "comprising" as used herein indicates the presence of features, steps, or operations, but does not exclude the presence or addition of one or more other features.

[0042] When using expressions such as "at least one of A, B, and C," the expression should generally be interpreted in accordance with the meaning commonly understood by a person skilled in the art (e.g., "a system having at least one of A, B, and C" should include, but is not limited to, a system having A alone, a system having B alone, a system having C alone, a system having A and B, a system having A and C, a system having B and C, and / or a system having A, B, and C, etc.). When using expressions such as "at least one of A, B, or C," the expression should generally be interpreted in accordance with the meaning commonly understood by a person skilled in the art.

[0043] Specifically, according to one embodiment of this application, a single molecular weight precision polyoxyethylene castor oil derivative is provided, having a structural formula as shown in formula (I) or formula (II):

[0044] Formula (I),

[0045] Formula (II), where x=y=z=4~100, w=4~100, a=1~10, b=0~5, c=1~10, d=0~5, e=1~10, f=0~5, g=0~5, h=1~10, and R is selected from any one of hydroxyl, methoxy, amino, carboxyl, and N-methylimidazole.

[0046] According to embodiments of this application, the values ​​of x, y, and z can be 4, 8, 11, 15, 20, 40, 60, 80, or 100, but are not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, they can be within a range consisting of any two values, such as 4~50, 45~100, etc.

[0047] According to embodiments of this application, the value of w can be 4, 9, 11, 16, 20, 50, 70, 90, or 100, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within a range consisting of any two values, such as 4~60, 50~100, etc.

[0048] According to embodiments of this application, the value of 'a' can be 1, 3, 5, 7, 9, or 10, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be a range consisting of any two values, such as 1~5, 4~10, etc.

[0049] According to embodiments of this application, the value of b can be 0, 1, 2, 3, 4, or 5.

[0050] According to embodiments of this application, the value of c can be 1, 2, 3, 4, 5, or 6, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be a range consisting of any two values, such as 1~6, 3~10, etc.

[0051] According to embodiments of this application, the value of d can be 0, 1, 2, 3, 4, or 5.

[0052] According to embodiments of this application, the value of e can be 1, 3, 5, 8, 9, or 10, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be a range consisting of any two values, such as 1~6, 5~10, etc.

[0053] According to embodiments of this application, the value of f can be 0, 1, 2, 3, 4, or 5.

[0054] According to embodiments of this application, the value of g can be 0, 1, 2, 3, 4, or 5.

[0055] According to embodiments of this application, the value of e can be 1, 2, 3, 4, 5, 6, 7, 8, or 9, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be a range consisting of any two values, such as 1~7, 6~10, etc.

[0056] According to the embodiments of this application, by precisely controlling the length of the polyoxyethylene chain segments and the branched structure, the molecular weight and molecular configuration are uniformly controllable, overcoming the problems of large product quality fluctuations and difficulty in reproducing key physicochemical properties caused by the wide molecular weight distribution of commercial polyoxyethylene castor oil. This derivative has both water solubility and drug loading and solubilizing ability, and can form nanoparticles with uniform and stable particle size, improving the solubility of poorly soluble drugs. At the same time, its precise molecular structure effectively reduces the risks of hemolysis, allergy and cytotoxicity caused by free surfactants, and exhibits higher biosafety when administered by injection.

[0057] According to embodiments of this application, the molecular weight of the single molecular weight precision polyoxyethylene castor oil derivative is 250~10000.

[0058] According to embodiments of this application, the molecular weight of a single-molecular-weight polyoxyethylene castor oil derivative can be 250, 500, 1000, 2000, 3000, 5000, 8000, or 10000, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within a range consisting of any two values, such as 250~6000, 4500~10000, etc.

[0059] According to the embodiments of this application, by limiting the molecular weight to the range of 250 to 10,000, precise control of molecular weight distribution is achieved, effectively overcoming the problems of large batch differences and uncontrollable quality caused by the uneven molecular weight of traditional polyoxyethylene castor oil. As a result, it exhibits consistent physicochemical properties and biological activity in the application of anti-tumor drugs, and can effectively improve the stability and safety of the formulation.

[0060] According to embodiments of this application, the single molecular weight precision polyoxyethylene castor oil derivative has the structure shown in formulas (I1) to (I9) or (II1) to (II8):

[0061] Equation (I1)

[0062] Equation (I2)

[0063] Equation (I3)

[0064] Equation (I4)

[0065] Equation (I5)

[0066] Equation (I6)

[0067] Equation (I7)

[0068] Equation (I8)

[0069] Equation (I9)

[0070] Formula (II1)

[0071] Formula (II2)

[0072] Equation (II3)

[0073] Formula (II4)

[0074] Formula (II5)

[0075] Equation (II6)

[0076] Formula (II7)

[0077] Formula (II8).

[0078] According to the embodiments of this application, by precisely controlling the length of the polyoxyethylene chain segments and the branched structure, the molecular weight and molecular configuration are uniformly controllable, overcoming the problems of large product quality fluctuations and difficulty in reproducing key physicochemical properties caused by the wide molecular weight distribution of commercial polyoxyethylene castor oil. This derivative has both water solubility and drug loading and solubilizing ability, and can form nanoparticles with uniform and stable particle size, improving the solubility of poorly soluble drugs. At the same time, its precise molecular structure effectively reduces the risks of hemolysis, allergy and cytotoxicity caused by free surfactants, and exhibits higher biosafety when administered by injection.

[0079] Furthermore, the preferred structure of the single molecular weight polyoxyethylene castor oil derivative shown in formula (I1) can be altered by double bonds and hydroxyl groups to make the tail hydrophobic chain structure more loosely arranged, reducing the tendency to crystallize. Hydrophobic drugs are more easily embedded in the micelle core, which can increase the drug loading capacity. At the same time, hydroxyl groups can also provide hydrogen bonding and dipole interactions with antitumor drug molecules, which are more conducive to the stability of drug encapsulation than simple hydrophilic-hydrophobic interactions.

[0080] According to embodiments of this application, a method for preparing a single molecular weight polyoxyethylene castor oil derivative is provided. The method for preparing the compound shown in formula (I) includes operations S101~S103:

[0081] In operation S101, the single molecular weight precise polyethylene glycol structure shown in formula (A) is subjected to a nucleophilic substitution reaction with the compound shown in formula (B), and after removing the protecting group, the compound shown in formula (C) is obtained.

[0082]

[0083] Formula (A) Formula (B)

[0084] In operation S102, the compound shown in formula (C) and the compound shown in formula (D) are subjected to an esterification reaction to obtain the compound shown in formula (E), wherein PG represents a protecting group;

[0085]

[0086] Equation (C) Equation (D)

[0087] In operation S103, the compound shown in formula (E) is deprotected to obtain the compound shown in formula (I).

[0088]

[0089] Equation (E)

[0090] According to embodiments of this application, prior to operation S101, the above preparation method further includes operation S100 for preparing a single molecular weight precise polyethylene glycol structure as shown in formula (A), specifically including sub-operations S1001 to S1002:

[0091] In suboperation S1001, one hydroxyl group of the single molecular weight precise polyethylene glycol structure shown in Formula S1 is protected with triphenylmethyl (Trt) to obtain the compound shown in Formula M1:

[0092]

[0093] S1

[0094] In suboperation S1002, the hydroxyl group of the compound shown in M1 is replaced with p-toluenesulfonyl chloride to obtain the single molecular weight precise polyethylene glycol structure shown in formula (A).

[0095]

[0096] M1

[0097] According to an embodiment of this application, prior to operation S102, the hydroxyl groups of ricinoleic acid are selectively protected using a tetrahydropyran (THP) protecting group to obtain the compound shown in formula (D).

[0098] According to embodiments of this application, the preparation method achieves precise preparation of the molecular structure of polyoxyethylene castor oil derivatives by introducing a specific single-molecular-weight polyethylene glycol structure as a building block and combining a protecting group strategy to carry out stepwise esterification and deprotection reactions.

[0099] According to the embodiments of this application, the solvent for the nucleophilic substitution reaction is selected from any one of N,N-dimethylformamide, tetrahydrofuran, and toluene, the reaction temperature is 0~60℃, and the reaction time is 4~72h; the solvent for the esterification reaction is selected from any one of dichloromethane, tetrahydrofuran, and toluene, the reaction temperature is 0~60℃, and the reaction time is 8~72h; the solvent for the deprotection reaction is selected from any one of dichloromethane and methanol, the reaction temperature is 0~60℃, and the reaction time is 2~72h.

[0100] According to embodiments of this application, the temperature for the nucleophilic substitution reaction can be 0°C, 10°C, 20°C, 30°C, 40°C, 50°C, or 60°C, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within a range consisting of any two values, such as 0~30°C, 40~60°C, etc.

[0101] According to embodiments of this application, the nucleophilic substitution reaction time can be 4h, 8h, 12h, 24h, 36h, 48h, or 72h, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within a range consisting of any two values, such as 4~32h, 24~72h, etc.

[0102] According to embodiments of this application, the temperature of the esterification reaction can be 0°C, 10°C, 25°C, 30°C, 45°C, 50°C, or 60°C, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within a range consisting of any two values, such as 0~40°C, 30~60°C, etc.

[0103] According to embodiments of this application, the esterification reaction time can be 8h, 12h, 24h, 32h, 50h, or 72h, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within a range consisting of any two values, such as 8~36h, 34~72h, etc.

[0104] According to embodiments of this application, the temperature for the deprotection reaction can be 0°C, 15°C, 25°C, 35°C, 45°C, 55°C, or 60°C, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within a range consisting of any two values, such as 0~50°C, 40~60°C, etc.

[0105] According to embodiments of this application, the reaction time for removing the protecting group can be 2h, 4h, 10h, 30h, 48h, or 72h, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within a range consisting of any two values, such as 2~30h, 20~72h, etc.

[0106] According to embodiments of this application, a mild synthetic process pathway was constructed by limiting the solvent system, temperature, and time for nucleophilic substitution, esterification, and deprotection reactions, ensuring high yield and high purity of polyoxyethylene castor oil derivatives with precise single molecular weight. Specifically, solvents with suitable polarity and chemical stability, such as N,N-dimethylformamide, tetrahydrofuran, and dichloromethane, were selected to effectively promote the dissolution of reactants. The reaction temperature was strictly controlled within a mild range of 0–60°C, ensuring sufficient reaction kinetics while minimizing the auto-oxidative degradation of polyoxyethylene segments at high temperatures, thus improving the chemical stability and molecular weight accuracy of the product. Combined with a staged reaction time design of 4–72 hours, each reaction step was carried out more thoroughly and uniformly, effectively avoiding the problem of molecular weight distribution broadening caused by local overheating or incomplete reaction.

[0107] According to embodiments of this application, a method for preparing a single molecular weight polyoxyethylene castor oil derivative is provided, wherein the preparation method of the compound shown in formula (II) includes operations S201~S202:

[0108] In operation S201, the single molecular weight precise polyethylene glycol structure shown in formula (F) is subjected to an esterification reaction with the compound shown in formula (G) to obtain the compound shown in formula (H), where PG represents a protecting group;

[0109]

[0110] Equation (F) Equation (G)

[0111] In operation S202, the protecting group of the compound shown in formula (H) is removed to obtain the compound shown in formula (II).

[0112]

[0113] Formula (H)

[0114] According to an embodiment of this application, prior to operation S102, the hydroxyl groups of ricinoleic acid are selectively protected using a tetrahydropyran (THP) protecting group to obtain the compound shown in formula (D).

[0115] According to embodiments of this application, by constructing ester bonds and introducing protecting groups, side reactions of unsaturated double bonds during esterification are effectively avoided, ensuring the precision and structural purity of molecular skeleton modification. The stepwise reaction design enhances the chemical stability of the product, thereby ensuring batch-to-batch uniformity of polymers with precise molecular weights, providing a reliable synthetic route for obtaining high-performance targeted delivery carriers.

[0116] According to the embodiments of this application, the solvent for the esterification reaction is selected from any one of dichloromethane, tetrahydrofuran, and toluene, the reaction temperature is 0~60℃, and the reaction time is 8~72h; the solvent for the deprotection reaction is selected from any one of dichloromethane and methanol, the reaction temperature is 0~60℃, and the reaction time is 2~72h.

[0117] According to embodiments of this application, the temperature of the esterification reaction can be 0°C, 5°C, 15°C, 35°C, 55°C, or 60°C, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within a range consisting of any two values, such as 0~50°C, 20~60°C, etc.

[0118] According to embodiments of this application, the esterification reaction time can be 8h, 12h, 24h, 32h, 50h, or 72h, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within a range consisting of any two values, such as 8~36h, 34~72h, etc.

[0119] According to embodiments of this application, the temperature for the deprotection reaction can be 0°C, 10°C, 15°C, 25°C, 35°C, 55°C, or 60°C, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within a range consisting of any two values, such as 0~20°C, 30~60°C, etc.

[0120] According to embodiments of this application, the reaction time for removing the protecting group can be 2h, 4h, 10h, 25h, 48h, or 72h, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within a range consisting of any two values, such as 2~40h, 25~72h, etc.

[0121] According to embodiments of this application, by limiting the esterification and deprotection reactions to mild conditions of 0–60°C and matching specific halogenated hydrocarbons or alcohol solvents, high-temperature degradation and side reactions of the polyoxyethylene segments are effectively suppressed, ensuring the structural integrity and high purity of the single molecular weight product. The optimized reaction time design balances conversion efficiency and process controllability, effectively improving the batch stability and chemical homogeneity of the product.

[0122] According to embodiments of this application, a nanoparticle is provided, which is assembled from a single molecular weight polyoxyethylene castor oil derivative through microfluidics, nano-flash delivery, mechanical dispersion, thin-film hydration, or ultrasonic dispersion.

[0123] According to embodiments of this application, the nanoparticles, relying on the structural uniformity of a single-molecular-weight polyoxyethylene castor oil derivative, eliminate the problem of wide particle size distribution caused by molecular weight differences in traditional polydisperse carriers. Through diverse assembly methods such as microfluidics and thin-film hydration, the derivative can form nanoparticles with uniform size and strong stability, effectively improving the encapsulation efficiency and sustained-release performance of hydrophobic drugs.

[0124] According to embodiments of this application, the average particle size of the nanoparticles of the single molecular weight precision polyoxyethylene castor oil derivative is 5~100nm, preferably 10~60nm, and the polydispersity index is <0.2.

[0125] According to embodiments of this application, the average diameter of the nanoparticles can be 5nm, 10nm, 20nm, 40nm, 60nm, 80nm, or 100nm, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within a range consisting of any two values, such as 5~60nm, 50~100nm, etc.

[0126] According to the embodiments of this application, the multidispersion index can be 0.03, 0.05, 0.08, 0.1, or 0.11, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0127] According to embodiments of this application, the limitation of particle size and polydispersity index more effectively leverages the advantage of the uniform structure of single molecular weight polyoxyethylene castor oil derivatives. A particle size range of 5–100 nm (particularly the preferred range of 10–60 nm) and a polydispersity index of less than 0.2 ensure the stability of the formulation in blood circulation, thereby improving drug utilization and therapeutic safety.

[0128] According to embodiments of this application, an application of nanoparticles in antitumor drug delivery is provided.

[0129] According to embodiments of this application, this application relies on the structural uniformity of a single molecular weight polyoxyethylene castor oil derivative and the size advantage of a preferred narrow distribution of 10~60nm, which can effectively match the delivery window of antitumor drugs: it can avoid premature interception by the reticuloendothelial system due to excessive particle size, and avoid loss through the renal pathway due to excessive particle size. Moreover, it can achieve efficient encapsulation and steady-state sustained release of hydrophobic antitumor drugs such as paclitaxel, thereby achieving the dual effects of improved efficacy and reduced toxic side effects.

[0130] According to the embodiments of this application, taking paclitaxel as an example, the concentration of paclitaxel in single molecular weight polyoxyethylene castor oil derivative nanoparticles is 0.2~5 mg / mL; while when the above nanoparticles are used, the concentration of paclitaxel is 0.1~2 mg / mL.

[0131] The present invention will be further described below with reference to specific embodiments. The raw materials used in this application are described as follows:

[0132] Triphenylmethyl chloride (TrtCl), dihydropyran (DHP), pyridine 4-methylbenzenesulfonic acid (PPTS), castor oil acid, and sodium hydride (NaH) were purchased from Anaiji and used directly. Triethylamine, sodium hydroxide (NaOH), p-toluenesulfonyl chloride (TsCl), dimethylaminopyridine (DMAP), dicyclohexylcarbodiimide (DCC), sodium chloride (NaCl), methanol, anhydrous sodium sulfate (Na2SO4), N,N-dimethylformamide (DMF), sodium bicarbonate (NaHCO3), tetrahydrofuran (THF), toluene (C7H8), dichloromethane (DCM), petroleum ether (PE), ethyl acetate (EA), ethanol, and diethyl ether (Et2O) were all purchased from Sinopharm Chemical Reagent Co., Ltd., and used directly unless otherwise specified. Anhydrous THF, MeCN, DCM, and toluene were obtained from the Pure-Solv 400 solvent purification system. Deionized water was prepared using the MILI-Q SP reagent water system (micropores), and the resistivity of the deionized water was 18.4 MΩcm. All other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd., and were used as is unless otherwise specified.

[0133] Example 1: Preparation of a single molecular weight polyoxyethylene castor oil derivative

[0134] The first step is to prepare the following single-protected, single-molecular-weight, precisely targeted polyethylene glycol monohydroxyl groups.

[0135]

[0136] Its characteristic is that one of the two hydroxyl groups of the compound shown in Formula 1 is selectively protected by triphenylmethyl (Trt).

[0137] Preparation method: Formula 1 (10.9 g, 20 mmol) was dissolved in 50 mL of anhydrous tetrahydrofuran, and triethylamine (5.0 g, 50 mmol) was added. Under a nitrogen atmosphere, triphenylmethyl chloride (7.0 g, 25 mmol) was dissolved in 10 mL of anhydrous tetrahydrofuran and added dropwise to the above system. The reaction was carried out at room temperature for 12 hours. The solvent was removed under reduced pressure, and the product was purified by column chromatography using petroleum ether (PE):ethyl acetate (EA) = 1:2 as the eluent to obtain 12.8 g of a colorless viscous liquid product (Formula 2) (yield 81%).

[0138]

[0139] The second step involves preparing the following single-protected, single-molecular-weight precision polyethylene glycol p-toluenesulfonate (Trt-OEG12-OTs).

[0140] Its characteristic is that the hydroxyl group of the compound shown in Formula 2 is replaced with p-toluenesulfonyl chloride to form p-toluenesulfonate.

[0141] Preparation method: The compound shown in Formula 2 (11.8 g, 15 mmol) was dissolved in 30 mL of anhydrous tetrahydrofuran. An aqueous solution of NaOH (2.6 g, 90 mmol dissolved in 10 mL of water) was added. Under a nitrogen atmosphere, p-toluenesulfonyl chloride (8.6 g, 45 mmol) was dissolved in 10 mL of anhydrous tetrahydrofuran and added dropwise to the above system under ice bath conditions. The reaction was carried out at room temperature for 12 hours. The solvent was removed under reduced pressure, and 200 mL of EA was added. Extraction was performed using EA and water (3 × 100 mL). The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product was purified by column chromatography using petroleum ether (PE):ethyl acetate (EA) = 2:1 as the eluent, yielding 12.6 g of a white solid product (Formula 3) (yield 89%).

[0142] Figure 1 This is the mass spectrum of the compound of formula 3 prepared in Example 1 of this application. According to... Figure 1 As can be seen, the compound shown in Formula 3 was obtained through verification.

[0143] The third step is to prepare the following three-armed, end-protected, single-molecular-weight precision polyethylene glycol (3arms-OEG12-Trt).

[0144]

[0145] Its characteristic is that the compound shown in Formula 3 undergoes a nucleophilic substitution reaction with the three hydroxyl groups of glycerol under basic conditions to generate a three-arm branched compound of Formula 4 with glycerol as the connecting point.

[0146] Preparation method: The compound shown in Formula 3 (12.5 g, 13.3 mmol) and glycerol (0.37 g, 4 mmol) were mixed in a flask and dehydrated three times by azeotropic treatment with toluene (30 mL × 3). Under a nitrogen atmosphere, 40 mL of anhydrous tetrahydrofuran was added as solvent. NaH (60% by mass dispersed in mineral oil, 0.64 g, 16 mmol) was added under ice bath conditions. The reaction was carried out at 50 °C for 12 hours. After the reaction was completed, NaH was quenched by slowly adding water (3 mL), the solvent was removed under reduced pressure, 200 mL of dichloromethane (DCM) was added, and the mixture was extracted with DCM and water (3 × 100 mL). The organic phase was dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the mixture was purified by column chromatography using petroleum ether (PE):ethyl acetate (EA) = 1:5 as the eluent to give 7.4 g of white solid product (Formula 4) (yield 77%).

[0147] Figure 2 This is the mass spectrum of the compound of formula 4 prepared in Example 1 of this application. According to... Figure 2 As can be seen, the compound shown in Formula 4 was verified, and its mass spectrometry characterization is as follows. Figure 2 The figure shows a precise single molecular weight.

[0148] The fourth step is to prepare the following single-molecular-weight polyethylene glycol with three-armed branched terminal hydroxyl groups (3arms-OEG12-OH).

[0149]

[0150] Its characteristic is that the triphenylmethyl group (Trt) at the end of each branched chain is removed using acid.

[0151] Preparation method: Dissolve Formula 4 (7.2 g, 3.0 mmol) in 20 mL of methanol (MeOH), stir well, add hydrochloric acid (4M dioxane hydrochloride solution, 1 mL, 4.0 mmol), and react for 4 hours. Remove the solvent under reduced pressure, add 200 mL of water, and extract with EA and water (100 mL × 3) to remove the removed Trt. Then extract with DCM and water (250 mL × 3) to extract the product into DCM. Combine the organic phases, dry with anhydrous sodium sulfate, remove the solvent under reduced pressure, and obtain 4.8 g of white solid product (Formula 5) (yield 95%).

[0152] Step 5: Prepare ricinoleic acid with tetrahydropyran (THP) protected hydroxyl groups.

[0153]

[0154] Its characteristic is that it uses a tetrahydropyran (THP) protecting group to selectively protect hydroxyl groups.

[0155] Preparation method: Formula 6 (10.5 g, 35 mmol) and PPTS (0.88 g, 3.5 mmol) were dissolved in 60 mL of anhydrous DCM. Under nitrogen protection, tetrahydropyran (THP) (2.9 g, 35 mmol) was dissolved in 10 mL of anhydrous DCM. The mixture was slowly added dropwise to the system under ice bath conditions, and the reaction was stirred for 8 hours. Water extraction (40 mL × 3) was performed. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product was purified by column chromatography using petroleum ether (PE):ethyl acetate (EA) = 10:1 as the eluent to obtain 9.4 g of a colorless viscous liquid product (Formula 7) (yield 70%).

[0156] Step 6: THP-protected ricinoleic acid and the product from step 4, 3arms-OEG12-OH, are esterified.

[0157]

[0158] Its characteristic is that the hydroxyl groups at the ends of the three arms undergo esterification with the carboxyl groups of ricinoleic acid.

[0159] Preparation method: Formula 5 (3 g, 1.8 mmol), Formula 7 (2.3 g, 6 mmol), and DMAP (44 mg, 0.36 mmol) were azeotropically dehydrated with toluene (20 mL × 3) under nitrogen protection. 20 mL of anhydrous DCM was added to dissolve the solutions. DCC (1.03 g, 5 mmol) was dissolved in 5 mL of anhydrous DCM and added to the system. The reaction was carried out at room temperature for 24 hours. The solvent was removed under reduced pressure, and the solution was purified by column chromatography using petroleum ether (PE):ethyl acetate (EA) at a ratio of 10:1 as the eluent, yielding 3.9 g of a colorless, viscous liquid product (Formula 8) (yield 78%).

[0160] Step 7: Remove THP protection from the side chain hydroxyl groups of ricinoleic acid.

[0161]

[0162] Its characteristic is that it uses a weak acid to remove THP protection without affecting the ester bond.

[0163] Preparation method: Formula 8 (2.7 g, 1 mmol) was dissolved in 20 mL of anhydrous methanol, and copper sulfate (0.8 g, 5 mmol) was added to the solution. The mixture was stirred for 48 hours. The copper sulfate was removed by filtration, and the solvent and protecting residue byproducts were removed under reduced pressure to obtain 2.4 g of a colorless viscous product (Formula 9) (yield 95%).

[0164] Figure 3 The mass spectrum of the compound of Formula 9 prepared in Example 1 of this application; Figure 4 The image shows the 1H NMR spectrum of the compound of Formula 9 prepared in Example 1 of this application. Figure 3 and Figure 4 As can be seen, the compound shown in Formula 9 was obtained through verification.

[0165] Comparative Example 1:

[0166] The commercially available polyoxyethylene castor oil used in Comparative Example 1 was analyzed by mass spectrometry. Figure 5 This is the mass spectrum of commercial polyoxyethylene castor oil, Comparative Example 1 of this application. According to... Figure 5 It can be seen that commercial polyoxyethylene castor oil has a wide molecular weight distribution, ranging from 1000 to 3500. The main peak molecular weight is 2331.927.

[0167] Further, gel permeation chromatography (THF phase GPC) using tetrahydrofuran (THF) as the mobile phase (eluent) was used to detect the commercial polyoxyethylene castor oil of Comparative Example 1 and the single molecular weight precision polyoxyethylene castor oil derivative of Example 1.

[0168] Figure 6 This is a gel permeation chromatogram of polyoxyethylene castor oil from Comparative Example 1 and Example 1 of this application. According to... Figure 6 It can be seen that the polydispersity index of commercial polyoxyethylene castor oil is higher than that of the single molecular weight polyoxyethylene castor oil derivative synthesized in Example 1 above.

[0169] Example 2: Construction of precise polyoxyethylene castor oil derivative nanoparticles with a single molecular weight

[0170] Weigh paclitaxel: Accurately weigh 12.0 mg of paclitaxel (PTX).

[0171] Dissolving paclitaxel: Dissolve the weighed paclitaxel in 1.0 mL of anhydrous ethanol. Ensure the paclitaxel is completely dissolved. Paclitaxel dissolves slowly in ethanol.

[0172] Add the single molecular weight precise polyoxyethylene castor oil derivative prepared in Example 1: After paclitaxel is completely dissolved in ethanol, add 1.0 mL of the single molecular weight precise polyoxyethylene castor oil derivative at a volume ratio of 1:1. Mix and shake thoroughly to form a transparent and homogeneous concentrate.

[0173] Flash precipitation: Each concentrate was rapidly injected into 0.9% physiological saline (4.0 mL) at 25 °C and stirred vigorously to obtain nanoparticles with an initial PTX concentration of 2.0 mg / mL.

[0174] Dialysis: Freshly prepared nanoparticles were transferred to a dialysis bag (MWCO=500Da) and dialyzed with 0.9% saline for 2 hours to reduce residual ethanol content. After dialysis, the nanoparticles prepared from a single molecular weight, precisely polyoxyethylene castor oil derivative were named PTX@p-Taxol (abbreviated as p-Taxol) and diluted to a PTX concentration of 0.5 mM (0.429 g / L) before use.

[0175] Comparative Example 2:

[0176] The preparation process of Comparative Example 2 is largely the same as that of Example 2. The difference is that in Comparative Example 2, the single molecular weight precise polyoxyethylene castor oil derivative is replaced with the commercial polyoxyethylene castor oil of Example 1 to prepare commercial polyoxyethylene castor oil nanoparticles, namely PTX@Taxol (abbreviated as Taxol).

[0177] The nanoparticles prepared in Example 2 and Comparative Example 2 were subjected to dynamic light scattering (DLS) to measure particle size and transmission electron microscopy (TEM) to measure particle size.

[0178] Figure 7 This is a particle size distribution diagram of the single molecular weight polyoxyethylene castor oil derivative nanoparticles prepared in Example 2 of this application, where f(D) h D represents the normalized particle size distribution function, reflecting the relative probability of particle size occurrence. h The particle size represented by μ2 / I 2 Represents a high degree of dispersion index; Figure 8 This is a particle size distribution diagram of the commercial polyoxyethylene castor oil nanoparticles prepared in Comparative Example 2 of this application, where f(D) h D represents the normalized particle size distribution function, reflecting the relative probability of particle size occurrence. h The particle size represented by μ2 / I 2 This represents the dispersion index.

[0179] according to Figure 7 and Figure 8 It can be seen that the average particle size of PTX@p-Taxol obtained from the single-molecular-weight precision polyoxyethylene castor oil derivative is 42 nm, and the polydispersity index is 0.108. The average particle size of PTX@Taxol obtained from commercial polyoxyethylene castor oil is 35 nm, and the polydispersity index is 0.169. The larger particle size and lower polydispersity index of PTX@p-Taxol compared to PTX@Taxol indicate that the single-molecular-weight precision polyoxyethylene castor oil derivative nanoparticles have a narrower particle size distribution.

[0180] Application Example 1: Stability evaluation and cytotoxicity study of PTX@p-Taxol obtained in Example 2 and PTX@Taxol obtained in Comparative Example 2.

[0181] 1. Stability Evaluation

[0182] 1.0 mM PTX@Taxol or PTX@p-Taxol dispersions (PBS buffer, pH 7.4, PTX concentration 0.5 mM) were left to stand at room temperature. The particle size and polydispersity index of the nanoparticles were measured using dynamic light scattering (DLS) at four time points: 0 days, 5 days, 10 days, and 14 days.

[0183] Figure 9 This is a graph showing the results of different nanoparticle sizes and polydispersity indices used in Application Example 1 of this application, where D... h The particle size represented by μ2 / I 2 This represents the dispersion index.

[0184] according to Figure 9 As can be seen, PTX@p-Taxol remained stable throughout the four test time points, with its particle size and polydispersity index remaining essentially unchanged. However, after 5 days, PTX@Taxol particles began to slowly aggregate, resulting in decreased stability, and the particle size and polydispersity index started to increase. After 10 days, both the particle size and polydispersity index of PTX@Taxol increased significantly. The stability evaluation results show that the particle stability of PTX@p-Taxol was significantly higher than that of PTX@Taxol during the test period.

[0185] 2. Cytotoxicity studies

[0186] The cultured 4T1 cells (mouse breast cancer cells) and 3T3 cells (mouse embryonic fibroblasts) were added to four 96-well plates, with 100 μL of DMEM medium added to each well and approximately 5000 cells. The plates were then cultured at 37°C and 5% CO2 for 24 hours.

[0187] Replace with new DMEM culture medium containing different concentrations of the assemblies PTX@p-Taxol and PTX@Taxol (corresponding to drug concentrations of 0, 1.5625, 3.125, 6.25, 12.5, 25, and 50 μM, respectively) and a blank control. There are 6 groups of each concentration. Incubate at 37°C and 5% CO2 for 48 hours.

[0188] Add 10 μL of CCK-8 solution to each well of a 96-well plate, incubate at 37°C in a 5% CO2 environment for 4 hours, and finally measure the absorbance at 450 nm using a microplate reader. The cytotoxicity results are attached. Figure 9 and 10 As shown.

[0189] Figure 10 The graph shows the cytotoxicity results of different nanoparticles used in Application Example 1 of this application against 4T1 tumor cells; Figure 11 This is a graph showing the cytotoxicity results of different nanoparticles used in Application Example 1 of this application on normal 3T3 cells.

[0190] according to Figure 10 and Figure 11 It can be seen that for 4T1 tumor cells, at all the concentrations used in the experiment, the PTX@p-Taxol group showed lower cell survival and greater toxicity, while the PTX@Taxol group showed higher cell survival and lower toxicity. For normal 3T3 cells, at all the concentrations used in the experiment, the PTX@p-Taxol and PTX@Taxol groups showed comparable cell survival and no significant difference in toxicity. This indicates that, under conditions of comparable toxicity to normal cells, PTX@p-Taxol exhibits better killing ability against tumor cells compared to PTX@Taxol.

[0191] Application Example 2: In vitro paclitaxel (PTX) release assay

[0192] 1.0 mL of the PTX@Taxol dispersion obtained in Comparative Example 2 or the PTX@p-Taxol dispersion obtained in Example 2 (PBS buffer, pH 7.4, PTX concentration 0.5 mM) was placed in a dialysis tube (MWCO = 3500 Da), then immersed in 20 mL of PBS medium and gently stirred at 25 °C. During the experiment, the external buffer was removed and replaced with fresh medium at predetermined time points. At each predetermined time point, 1 mL of buffer sample was taken and diluted 2-fold with deionized water. The drug concentration was quantitatively analyzed by HPLC according to the standard curve.

[0193] Figure 12 The graph shows the results of in vitro paclitaxel (PTX) release assays using different nanoparticles in Application Example 2 of this application.

[0194] according to Figure 11 It can be seen that the release rate of PTX in both PTX@Taxol and PTX@p-Taxol decreases over time. However, the drug release rate of PTX@p-Taxol is significantly lower than that of PTX@Taxol, indicating that PTX@p-Taxol is more stable and less prone to leakage compared to PTX@Taxol. During the 120-hour test period, the drug release rate of PTX@p-Taxol reached equilibrium at around 4%, while the drug release rate of PTX@Taxol reached 14% and continued to increase.

[0195] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of this application. It should be understood that the above descriptions are merely specific embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A single molecular weight polyoxyethylene castor oil derivative having a structure as shown in formula (I) or formula (II): Equation (I), Equation (II), where, x=y=z=4~100, w=4~100, a=1~10, b=0~5, c=1~10, d=0~5, e=1~10, f=0~5, g=0~5, h=1~10, and R is selected from any one of hydroxyl, methoxy, amino, carboxyl, and maleimide.

2. The single molecular weight precision polyoxyethylene castor oil derivative according to claim 1, wherein, The molecular weight of the single-molecular-weight polyoxyethylene castor oil derivative is 250~10000.

3. The single molecular weight precision polyoxyethylene castor oil derivative according to claim 1 or 2, wherein, The single molecular weight precision polyoxyethylene castor oil derivative has the structure shown in formulas (I1) to (I9) or (II1) to (II8): Equation (I1) Equation (I2) Equation (I3) Equation (I4) Equation (I5) Equation (I6) Equation (I7) Equation (I8) Equation (I9) Equation (II1) Equation (II2) Equation (II3) Equation (II4) Formula (II5) Equation (II6) Formula (II7) Formula (II8).

4. A method for preparing a single molecular weight precision polyoxyethylene castor oil derivative as described in any one of claims 1 to 3, wherein, The preparation method of the compound shown in formula (I) includes: The compound shown in formula (A) with a single molecular weight of precisely polyethylene glycol was subjected to a nucleophilic substitution reaction with the compound shown in formula (B), and after removing the protecting group, the compound shown in formula (C) was obtained. Formula (A) Formula (B) The compound shown in formula (C) and the compound shown in formula (D) are subjected to esterification to obtain the compound shown in formula (E), wherein PG represents a protecting group; Equation (C) Equation (D) The compound shown in formula (E) was deprotected to obtain the compound shown in formula (I); Equation (E).

5. The preparation method according to claim 4, wherein, The solvent for the nucleophilic substitution reaction is selected from any one of N,N-dimethylformamide, tetrahydrofuran, and toluene; the reaction temperature is 0~60℃; and the reaction time is 4~72h. The solvent for the esterification reaction is selected from any one of dichloromethane, tetrahydrofuran, and toluene, the reaction temperature is 0~60℃, and the reaction time is 8~72h; The solvent for the deprotection reaction is selected from either dichloromethane or methanol, the reaction temperature is 0~60℃, and the reaction time is 2~72h.

6. A method for preparing a single molecular weight precision polyoxyethylene castor oil derivative as described in any one of claims 1 to 3, wherein, The preparation method of the compound shown in formula (II) includes: The single molecular weight polyethylene glycol structure shown in formula (F) is esterified with the compound shown in formula (G) to obtain the compound shown in formula (H), where PG represents a protecting group; Equation (F) Equation (G) The compound shown in formula (H) is deprotected to obtain the compound shown in formula (II); Formula (H).

7. The preparation method according to claim 6, wherein, The solvent for the esterification reaction is selected from any one of dichloromethane, tetrahydrofuran, and toluene, the reaction temperature is 0~60℃, and the reaction time is 8~72h; The solvent for the deprotection reaction is selected from either dichloromethane or methanol, the reaction temperature is 0~60℃, and the reaction time is 2~72h.

8. A nanoparticle, wherein, The nanoparticles are assembled from single molecular weight polyoxyethylene castor oil derivatives as described in any one of claims 1 to 3 by means of microfluidics, nano-flash delivery, mechanical dispersion, thin film hydration, or ultrasonic dispersion.

9. The nanoparticles according to claim 8, wherein, The nanoparticles of the single molecular weight precision polyoxyethylene castor oil derivative have an average particle size of 5~100nm, preferably 10~60nm, and a polydispersity index <0.

2.

10. The application of the nanoparticles as described in claim 9 in the delivery of antitumor drugs.

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