Preparation method and application of gambogic acid self-assembled micelle nanoparticles

Abstract

The invention discloses a preparation method and application of esterase sensitive gambogic acid self-assembled micelle nanoparticles. Gambogic acid and poloxamer 188 are used as raw materials of the self-assembled micelle nanoparticle, and GA and P188 form an ester bond through esterification reaction, so that a gambogic acid self-assembled micelle nanoparticle precursor is formed. The gambogic acid micelle nanoparticles can be formed through self-assembly in an aqueous solution, the particle size is uniform, the average particle size is about 58 nm, the polydispersity index is 0.203, and it is indicated that the micelle nanoparticles have good monodispersity. In-vitro release studies show that the prepared gambogic acid self-assembled micelle nanoparticles have esterase responsiveness and are expected to realize targeted release of gambogic acid at tumor sites, and the anti-tumor effect is improved. The preparation method of the gambogic acid self-assembled micelle nanoparticle GA-P188 provided by the invention is simple in operation steps, relatively low in cost and good in tumor inhibition effect, and can have relatively high industrialization prospects.

Description

Technical Field

[0001] This invention relates to a method for preparing and applying gambogeylic acid self-assembled micelle nanoparticles, belonging to the field of pharmaceutical synthesis technology. Background Technology

[0002] Gambogic acid is a lipid-soluble polycyclic xanthone derivative with the molecular formula C2. 38 H 44 O8, with a relative molecular mass of 628.75, is gambogeylic acid (GA). At room temperature, it is a deep yellow to orange-red crystal, insoluble in water, slightly soluble in polar solvents such as methanol and ethanol, and readily soluble in chloroform, dichloromethane, dimethyl sulfoxide (DMSO), acetone, and ethyl acetate. Its solubility in pure GA decreases significantly with increasing purity. GA is a potent natural inhibitor of tumor cell proliferation, exhibiting antitumor activity tens to hundreds of times greater than traditional chemotherapy drugs. Its core pharmacological effects include inducing tumor cell apoptosis, arresting the cell cycle, inhibiting angiogenesis, and suppressing tumor metastasis. Preclinical studies have shown significant therapeutic potential against various malignant tumors, including liver cancer, lung cancer, gastric cancer, and breast cancer, and it is considered a promising natural anticancer lead compound. Despite its excellent antitumor activity, GA's poor solubility in water and low bioavailability make its clinical application extremely challenging.

[0003] Drug-polymer conjugates are compounds formed by covalently linking drug molecules to a polymeric carrier. By binding drugs to polymeric carriers, drug solubility and loading capacity are improved, ensuring the drug reaches tumor tissue more effectively. Small molecule drugs, after conjugation to polymeric carriers, can autonomously assemble into nanomicelles. Due to their small size effect and surface / interface effects, their key advantages include: passive targeting, avoiding rapid renal clearance, and prolonging the drug's plasma half-life. Furthermore, depending on the properties of the polymeric carrier and the conjugation method, slow and sustained drug release can be achieved, maintaining effective therapeutic concentrations in tumor tissue for a longer period, thereby enhancing efficacy.

[0004] Poloxamer is a nonionic surfactant with a triblock copolymer structure composed of polyethylene oxide (PEO) and polypropylene oxide (PPO), with the general structural formula HO(C2H4O)a(C3H6O)b(C2H4O)aH, where the PEO segment is polyethylene oxide and the PPO segment is polypropylene oxide. Poloxamer 188 can be used as a carrier material to bind drugs through chemical bonding, achieving targeted delivery, increasing drug concentration in target tissues, and reducing toxicity to normal tissues.

[0005] Currently, many polymer-drug conjugates are mainly polyethylene glycol (PEG)-antitumor drug conjugates. However, their degradation products in vivo (such as PEG oxides) have certain toxicity, posing safety concerns. Poloxamer, on the other hand, has a relatively high safety profile, but there are no research reports on the use of poloxamer 188 in conjugates with the antitumor drug gamboge to exert antitumor effects. Summary of the Invention

[0006] The purpose of this invention is to provide a simple and low-cost method for preparing and applying gambogeylic acid self-assembled micelle nanoparticles (GA-P188). This invention grafts gambogeylic acid with poloxamer 188 via ester bonds, thereby improving the water solubility of gambogeylic acid and obtaining an amphiphilic gambogeylic acid prodrug capable of self-assembling into nanostructures in aqueous solution.

[0007] A self-assembled micelle nanoparticle of gambogeylic acid (GA-P188) has the following molecular structure: A method for preparing gambogeylic acid self-assembled micelle nanoparticles (GA-P188) involves using gambogeylic acid (GA) and poloxamer 188 (P188) as raw materials, and carrying out the reaction in a solvent in the presence of a condensing agent and a catalyst according to the reaction formula shown below: A method for preparing gambogeylic acid self-assembled micelle nanoparticles (GA-P188) includes the following steps: (1) Add gambogeylic acid (GA), poloxamer 188 (P188), condensing agent and catalyst to a reaction vessel, add solvent in air or inert gas atmosphere, react at a specific temperature to obtain reaction solution ①; (2) Transfer the reaction solution ① to a regenerated cellulose dialysis bag with a molecular weight cutoff of 3500 Da, place the dialysis bag in the solvent for dialysis, and change the dialysis solution every 4 hours. (3) After dialysis, freeze-dry the solution in the dialysis bag to remove excess solvent and obtain the GA-P188 precursor. (4) The above GA-P188 precursor was placed in water and dissolved by ultrasonication. After being filtered through a 0.45μm filter membrane, gambogeylic acid self-assembled micelle nanoparticles were obtained.

[0008] In step (1), the molar ratio of gambogeylic acid to poloxamer 188 is 0.5:1 to 5:1.

[0009] In step (1), the molar ratio of gamboge acid to condensing reagent is 0.2:1 to 1:1.

[0010] In step (1), the molar ratio of gamboge acid to catalyst is 1:1 to 20:1.

[0011] In step (1), the condensing agent is selected from at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, and N,N'-diisopropylcarbodiimide.

[0012] In step (1), the catalyst is selected from at least one of 4-dimethylaminopyridine, 1-hydroxybenzotriazole, and 4-dimethylaminopyridine N-oxide.

[0013] The solvent in step (1) is selected from at least one of dichloromethane, 1,2-dichloroethane, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, ethyl acetate, ethylene glycol dimethyl ether, chloroform, and toluene.

[0014] The amount of solvent used should be sufficient to meet the reaction requirements, and the preferred ratio of solvent to gamboge acid is 1 mmol: 5~50 mL.

[0015] The reaction temperature in step (1) is 0℃~100℃.

[0016] In step (2), the dialysis solvent is selected from at least one of dichloromethane, 1,2-dichloroethane, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, ethyl acetate, ethylene glycol dimethyl ether, chloroform, and toluene.

[0017] In a preferred embodiment of the present invention, in step (1), the ratio of gambogeylic acid, poloxamer 188, condensing agent and catalyst is 0.15 mmol : 0.1 mmol : 0.3 mmol : 0.03 mmol, and the volume of dichloromethane is 20 mL.

[0018] In a preferred embodiment of the present invention, the condensing agent in step (1) is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0019] In a preferred embodiment of the present invention, the catalyst in step (1) is 4-dimethylaminopyridine.

[0020] In a preferred embodiment of the present invention, the solvent in step (1) is dichloromethane.

[0021] In a preferred embodiment of the present invention, step (1) is carried out in an argon atmosphere.

[0022] In a preferred embodiment of the present invention, the reaction temperature of step (1) is 25 °C.

[0023] The present invention provides a method for preparing the self-assembled precursor of gambogey acid to form nanomicelles as described above: the gambogey acid self-assembled precursor powder obtained after freeze-drying is placed in water, and it can self-assemble to form micelle nanoparticles. After filtration through a filter membrane, gambogey acid self-assembled micelle nanoparticles are obtained.

[0024] This invention relates to the application of gambogeylic acid self-assembled micelle nanoparticles as antitumor drug carriers.

[0025] The beneficial effects of this invention are: This invention provides a method for preparing the self-assembled prodrug of gambogeylic acid described above. Using gambogeylic acid and poloxamer 188 as raw materials, the self-assembled prodrug of gambogeylic acid can be obtained through a simple reaction in the presence of condensing reagent and catalyst. The steps are simple, easy to operate, and low in cost, and have high industrialization prospects.

[0026] This invention provides a method for preparing self-assembled nanomicelles from the above-described gambogeylic acid precursor.

[0027] The results of the examples show that the gamboge acid self-assembly precursor provided by the present invention can self-assemble into micelles in aqueous solution, and has high solubility and stability in aqueous solution. Attached Figure Description

[0028] Figure 1 This is the NMR spectrum of the self-assembled precursor of gambogeylic acid; Figure 2 This is a particle size characterization diagram of gambogeylic acid self-assembled nanoparticles; Figure 3 This is a transmission electron microscope image of self-assembled gambogeylic acid nanoparticles; Figure 4 This is a differential scanning calorimetry curve of gamboge acid self-assembled nanoparticles; Figure 5 This is a diagram showing the CCK-8 cytotoxicity experiment results of gambogeylic acid self-assembled nanoparticles. Detailed Implementation

[0029] To make the technical problems solved, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and materials described are commercially available unless otherwise specified. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0030] Example 1 Preparation of the self-assembled precursor of gambogeylic acid. At room temperature, 100.0 mg (0.15 mmol) of gambogeylic acid, 840.0 mg (0.1 mmol) of poloxamer 188, 61.0 mg (0.31 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 3.7 mg (0.03 mmol) of 4-dimethylaminopyridine, and 5 mL of anhydrous dichloromethane were added sequentially to a 25 mL Schlenk flask under argon protection. The solution was reacted at 25 °C for 12 hours under argon atmosphere. After the reaction was complete, the reaction solution was transferred to a regenerated cellulose dialysis bag and dialyzed with dichloromethane as the dialysis solvent until the solution color no longer changed. The solution in the dialysis bag was then freeze-dried to obtain 761.4 mg of the self-assembled precursor of gambogeylic acid, with a yield of 84.33%. Figure 1 This is the NMR spectrum of the self-assembled precursor of gambogeylic acid prepared in Example 1.

[0031] Example 2 Except that the amount of gambogeylic acid used in Example 1 was changed to 63.0 mg (0.10 mmol), the same method as in Example 1 was followed to obtain a pre-assembly yield of 81.04% for gambogeylic acid.

[0032] Example 3 Except that the amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in Example 1 was changed to 38.3 mg (0.2 mmol), the same method as in Example 1 was followed to obtain a pre-assembly yield of 80.64% of gambogeylic acid.

[0033] Example 4 Except that the amount of 4-dimethylaminopyridine used in Example 1 was changed to 1.2 mg (0.01 mmol), the same method as in Example 1 was followed to obtain a pre-assembly yield of 80.50% of gambogeylic acid.

[0034] Example 5 Except for replacing the reaction solvent and dialysis solvent in Example 1 with anhydrous N,N-dimethylformamide, the same method as in Example 1 was followed to obtain a pre-assembly yield of 83.58% for gambogeylic acid.

[0035] Example 6 Except for replacing the reaction solvent and dialysis solvent in Example 1 with anhydrous dimethyl sulfoxide, the same method as in Example 1 was followed to obtain a pre-assembly yield of 84.00% for gambogeylic acid.

[0036] Example 7 The preparation and particle size testing of the self-assembled precursor of gambogeylic acid were carried out. Particle size testing was performed on Examples 1-6. A certain amount of lyophilized powder of the self-assembled precursor of gambogeylic acid obtained from Examples 1-6 was weighed and placed in an appropriate amount of water. After being completely dissolved by sonication, the powder was filtered through a filter membrane to obtain gambogeylic acid self-assembled micelle nanoparticles. Figure 2 The particle size test results for Examples 1-6 are as follows: the average particle sizes of the micelles are 58.66 nm, 78.63 nm, 66.66 nm, 66.89 nm, 93.79 nm, and 55.85 nm, respectively.

[0037] Example 8 Transmission electron microscopy (TEM) was used to analyze the pre-assembly of gambogeylic acid. The morphology of the micelles was observed using TEM. A GA-P188 micelle solution of a certain concentration was first prepared. A small amount of the nano-micelle solution was then dropped onto a copper mesh, which was subsequently dried in an oven at 37°C for 30 minutes. After air drying, the morphology of GA-P188 was observed using TEM. Figure 3 A is a schematic diagram of the micelles of GA-P188 in Example 8. Figure 3 B represents the TEM test results of Example 8. GA-P188 is nearly spherical and has a relatively uniform particle size.

[0038] Example 9 Differential scanning calorimetry (DSC) was used to analyze the pre-assembly of gambogeylic acid. Three samples were prepared for DSC analysis: GA (5 mg), P188 (5 mg), and GA-P188 (5 mg). Each sample was accurately weighed and sealed in an aluminum pan. The temperature was set between 25 and 200 °C, the heating rate was 15 ml / min, nitrogen was used as the protective gas, and the flow rate was 20 ml / min. Figure 4 The DSC test results for Example 9 show that GA and P188 raw materials have endothermic peaks at 72.2℃ and 58.4℃, respectively. Two peaks appeared simultaneously in the physical mixture. However, in the test curve of the gamboge acid self-assembled precursor (GA-P188), no peak of GA raw material appeared, and only one peak appeared at 52.1℃. Compared with the endothermic peak of P188 raw material at 58.4℃, the peak of P188 raw material was significantly shifted, which verifies the successful synthesis of gamboge acid self-assembled precursor.

[0039] Example 10 CCK-8 cytotoxicity assays were performed on the pre-assembly drug of gambogeylic acid. The concentration was 1×10⁻⁶. 4Cells were added to 96-well plates at 100 μL / well and cultured for 24 h. Different concentrations of GA-P188 samples (1.6, 0.8, 0.6, 0.5, 0.2, 0.1, and 0.05 μg / mL) were prepared, with a blank control group using fresh DMEM complete medium. Each sample was prepared in 5 replicates. The culture medium from the previous day was precipitated, and the samples were added to 96-well plates for co-culture with B16F10 melanoma cells according to the experimental design. After 24 h of co-culture, 100 μL of 10% CCK-8 was added to the 96-well plates. After incubation at 37°C for 1.5 h, the absorbance of each well was measured at 490 nm and compared with the blank control group. Cell viability was determined according to the formula. Figure 5 The results of the CCK-8 cytotoxicity experiment in Example 10 show that GA-P188 has an IC50 of 0.31 μg / mL against melanoma, indicating a good tumor inhibition effect.

[0040] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A self-assembled micelle nanoparticle GA-P188 of gambogeylic acid, characterized in that: The GA-P188 has the following molecular structure: 。 2. The preparation method of GA-P188 according to claim 1 specifically includes the following steps: (1) Add gambogeylic acid GA, poloxamer 188 (P188), condensing reagent and catalyst to the reaction vessel, add solvent in air or inert gas atmosphere, react at a specific temperature to obtain reaction solution ①; (2) Transfer the reaction solution ① to a regenerated cellulose dialysis bag with a molecular weight cutoff of 3500 Da, and dialyze once every 4 hours. Place the dialysis bag in a solvent and dialyze until the liquid is clear and there is no yellow color. (3) After dialysis, freeze-dry the solution in the dialysis bag to remove excess solvent and obtain the GA-P188 precursor of gambogeylic acid self-assembled micelle nanoparticles. (4) The above-mentioned gambogey acid nanoparticle GA-P188 precursor was placed in water, dissolved by ultrasonication, and filtered through a 450μm filter membrane to obtain gambogey acid self-assembled micelle nanoparticles.

3. The method for preparing the GA-P188 precursor of gambogeylic acid self-assembled micelle nanoparticles as described in claim 2, characterized in that: In step (1), the molar ratio of gambogeylic acid to poloxamer 188 is 0.5:1 to 5:1; in step (1), the molar ratio of gambogeylic acid to condensing agent is 0.2:1 to 1:1; in step (1), the molar ratio of gambogeylic acid to catalyst is 1:1 to 20:

1.

4. The method for preparing the GA-P188 precursor of gambogeylic acid self-assembled micelle nanoparticles as described in claim 2, characterized in that: In step (1), the condensing agent is selected from at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, and 4-dimethylaminopyridine.

5. The method for preparing the GA-P188 precursor of gambogeylic acid self-assembled micelle nanoparticles as described in claim 2, characterized in that: The catalyst in step (1) is selected from one of 4-dimethylaminopyridine, 1-hydroxybenzotriazole, and 4-dimethylaminopyridine N-oxide.

6. The method for preparing the GA-P188 precursor of gambogeylic acid self-assembled micelle nanoparticles as described in claim 2, characterized in that: In step (1), the solvent is selected from one of dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, ethyl acetate, and ethylene glycol dimethyl ether, and the ratio of solvent volume to gamboge acid is 1 mmol: 5~50 mL.

7. The method for preparing the GA-P188 precursor of gambogeylic acid self-assembled micelle nanoparticles as described in claim 2, characterized in that: The reaction temperature in step (1) is 0℃~100℃.

8. The method for preparing the GA-P188 precursor of gambogeylic acid self-assembled micelle nanoparticles as described in claim 2, characterized in that: In step (2), the dialysis solvent is selected from at least one of dichloromethane, 1,2-dichloroethane, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, ethyl acetate, ethylene glycol dimethyl ether, chloroform, and toluene.

9. The application of the gambogeylic acid self-assembled micelle nanoparticles GA-P188 as an antitumor drug carrier as described in claim 1.

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