A method for improving liposome encapsulation efficiency by modifying cholesterol complex prodrug

Abstract

The present invention discloses a method for improving the liposome encapsulation efficiency by modifying a cholesterol composite prodrug. Specifically, the drug curcumin and vitamin E succinate are first reacted with cholesterol to obtain a cholesterol-curcumin complex and a cholesterol-vitamin E succinate complex. The two obtained complexes are then uniformly mixed with a lipid and then prepared by thin film hydration to obtain drug-loaded liposomes. The present invention utilizes the cholesterol composite prodrug method to improve the low solubility of curcumin and vitamin E succinate in liposomes, improve the encapsulation efficiency of the two drugs in liposomes, and provide a new approach for the preparation of liposomes containing poorly soluble compounds.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine and relates to a method for improving liposome encapsulation efficiency by modifying cholesterol composite prodrug. Background Art

[0002] Improving the in vivo bioavailability of poorly soluble compounds has always been a key topic in pharmaceutical research. However, many poorly soluble compounds are not only poorly soluble in water but also lipid-insoluble, meaning they are also difficult to dissolve in commonly used solvents under physiological conditions. This poses significant challenges to drug dosage form design and application.

[0003] As a drug delivery system, liposomes can penetrate the interstitial spaces between capillary epithelial cells and enter tumor tissue when drugs are delivered to tumor blood vessels, thereby achieving a certain degree of tumor targeting. The passive targeting properties of liposome-based drug delivery systems can alter drug distribution in the body, both improving efficacy and reducing toxicity, making them ideal vehicles for increasing the drug loading capacity of poorly soluble compounds. The use of liposomes solves the problem of low solubility of poorly soluble drugs, reduces toxic side effects, and simultaneously achieves better targeted distribution characteristics.

[0004] Although liposomes can effectively increase the solubility of poorly soluble compounds, the encapsulation efficiency of most liposome-based drug delivery systems still fails to reach the 85% required for marketing approval as an injectable solution. To further improve encapsulation efficiency, researchers have proposed several approaches, including: modifying preparation methods (e.g., preparing proliposomes); adding surfactants to the formulation to create vesicles; and adding surfactants and scaffold materials to create liposome-based solid nanoparticles. However, these approaches still have limitations. For certain lipid- and water-insoluble compounds, significant increases in encapsulation efficiency remain difficult, and the addition of surfactants to the formulation can cause significant toxic side effects. Therefore, improving liposome encapsulation efficiency while minimizing potential toxic side effects remains a key challenge. Summary of the Invention

[0005] Based on the problems existing in the above-mentioned prior art, the present invention provides a method for improving the liposome encapsulation efficiency by modifying the cholesterol complex prodrug, so as to improve the encapsulation efficiency and stability of the drug in the liposome and obtain drug-loaded liposomes with high encapsulation efficiency.

[0006] In order to achieve the purpose, the present invention adopts the following technical solutions:

[0007] A method for improving liposome encapsulation efficiency by modifying cholesterol-complexed prodrugs is characterized by first reacting the drugs curcumin and vitamin E succinate with cholesterol to obtain a cholesterol-curcumin complex and a cholesterol-vitamin E succinate complex, then uniformly mixing the two complexes with lipids and then preparing drug-loaded liposomes through thin film hydration. Specifically, the method comprises the following steps:

[0008] Step 1: Preparation of cholesterol-curcumin complex (CHO-CUR) by solvent evaporation method

[0009] Curcumin (CUR) and cholesterol (CHO) were dissolved in acetone and stirred for reaction, and then the acetone was removed by rotary evaporation and vacuum drying to obtain cholesterol-curcumin complex (CHO-CUR).

[0010] Preferably, the molar ratio of curcumin to cholesterol is 1:2, the stirring reaction temperature is 40-60° C., and the reaction time is 1-3 hours.

[0011] Step 2: Synthesis of cholesterol-vitamin E succinate complex (CHO-VES) by esterification reaction

[0012] Vitamin E succinate (VES) is added to an organic solvent, followed by the addition of a catalyst and cholesterol, and the mixture is stirred at room temperature under nitrogen protection. After the reaction is completed, the crude product is washed and rotary evaporated to obtain a crude product, which is then purified to obtain a cholesterol-vitamin E succinate complex (CHO-VES).

[0013] Preferably, the molar ratio of vitamin E succinate and cholesterol is 1.2:1, the catalysts used are 4-dimethylaminopyridine DMAP and 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC, and the mass of DMAP accounts for 10-15% of the total mass of vitamin E succinate and cholesterol, and the mass of EDC accounts for 15-20% of the total mass of vitamin E succinate and cholesterol.

[0014] Preferably, the organic solvent is anhydrous dichloromethane.

[0015] Preferably, the washing is performed sequentially with a 5% citric acid aqueous solution and saturated saline.

[0016] Preferably, the purification is carried out by column chromatography using petroleum ether and ethyl acetate.

[0017] Step 3: Prepare drug-loaded liposomes by thin film hydration

[0018] 1-10 parts of a cholesterol-curcumin complex, 1-10 parts of a cholesterol-vitamin E succinate complex, 1-20 parts of cholesterol, 1-20 parts of polyethylene glycol-distearoyl phosphatidylethanolamine, and 10-100 parts of soybean lecithin are dissolved in a mixture of chloroform and methanol, mixed uniformly by ultrasonication, and then the solvent is removed by rotary evaporation to obtain a uniform lipid film. A phosphate buffer solution is added, and the film is heated and stirred to fully hydrate the film. The hydrated solution is subjected to ultrasonication and filtration to obtain the target drug-loaded liposome solution.

[0019] Preferably, the heating and stirring temperature is 60-65° C. and the time is 10-30 min; the ultrasonic time is 10-30 min; and the filtration is performed through a 0.45 μm cellulose membrane.

[0020] Compared with the prior art, the beneficial effects of the present invention are embodied in:

[0021] The present invention utilizes cholesterol to react with curcumin (CUR) or vitamin E succinate (VES), which can improve the solubility of the two drugs in the liposomes during the subsequent liposome preparation process, thereby greatly improving the encapsulation rate and stability of the two drugs in the liposomes.

[0022] The method of the invention does not use a surfactant, thereby reducing the toxic and side effects of drugs.

[0023] The liposome drug-carrying system of the present invention can improve the drug encapsulation rate while ensuring the drug efficacy, reduce the amount of liposomes used, and improve the safety and efficacy of clinical application.

[0024] The method of the present invention is applicable to poorly soluble compounds, can significantly improve the encapsulation efficiency of poorly soluble compounds (especially lipid-water insoluble compounds), and provides a new idea for the preparation of liposomes of poorly soluble compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the synthesis of CHO-CUR;

[0026] Figure 2 For cholesterol 1 H NMR spectrum;

[0027] Figure 3 For curcumin 1 H NMR spectrum;

[0028] Figure 4 is the CHO-CUR prepared in the example 1 H NMR spectrum;

[0029] Figure 5 Schematic diagram of the synthesis of CHO-VES;

[0030] Figure 6 is the CHO-VES prepared in the example 1 H NMR spectrum;

[0031] Figure 7 TEM image of the drug-loaded liposomes prepared in the examples;

[0032] Figure 8 This is a graph showing the encapsulation efficiency of the drug-loaded liposomes prepared in the examples. DETAILED DESCRIPTION

[0033] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process. However, the protection scope of the present invention is not limited to the following embodiment.

[0034] Example 1

[0035] In this example, drug-loaded liposomes were prepared according to the following steps:

[0036] Step 1: Prepare CHO-CUR

[0037] Curcumin (CUR) and cholesterol (CHO) were dissolved in an appropriate amount of acetone at a molar ratio of 1:2, stirred at 50°C for 2 h, and then the acetone was removed by rotary evaporation and vacuum drying to obtain cholesterol-curcumin complex (CHO-CUR). Figure 1 shown.

[0038] Cholesterol, curcumin and CHO-CUR were 1 H NMR test results are as follows Figure 2 、 Figure 3 and Figure 4 shown. Figure 4 The peaks at 6.79, 6.99, and 7.11 ppm correspond to hydrogen on the benzene ring and come from curcumin, and the peaks at 1.19, 1.25, 1.30, and 1.62 ppm correspond to hydrogen on the long-chain alkyl group and come from cholesterol, indicating that the CHO-CUR complex was successfully synthesized.

[0039] Step 2: Preparation of CHO-VES

[0040] Weigh 0.85 g of VES and add 30 mL of anhydrous dichloromethane. Add precisely weighed 0.19 g of EDC, 0.15 g of DMAP and 0.31 g of CHO in sequence (the molar ratio of VES to CHO is 1.2:1 at this time), and stir at room temperature under nitrogen protection for 12 hours. After the reaction is completed, wash with 5% citric acid aqueous solution and saturated brine three times each, and then remove the organic solvent by rotary evaporation to obtain crude CHO-VES. The crude CHO-VES was separated and purified by column chromatography using petroleum ether and ethyl acetate (volume ratio 3:1). After elution is completed, the product is rotary evaporated to remove the organic solvent, and the resulting solid is CHO-VES. The synthetic route is as follows. Figure 5 shown.

[0041] CHO-VES 1 The results of H NMR tests are as follows Figure 6 The peak at 2.08 ppm corresponds to hydrogen on the benzene ring and comes from vitamin E succinate. The peaks at 1.65 and 1.9 ppm correspond to hydrogen on the five-membered ring and come from cholesterol. In addition, the peak at 4.77 ppm corresponding to the hydroxyl group disappears, and the peak at 12.16 ppm corresponding to the carboxyl group disappears, indicating that cholesterol and vitamin E succinate have successfully undergone esterification reaction and CHO-VES has been successfully synthesized.

[0042] Step 3: Preparation of drug-loaded liposomes

[0043] 1 part of CHO-CUR complex, 1 part of CHO-VES complex, 2 parts of cholesterol, 2 parts of polyethylene glycol-distearoylphosphatidylethanolamine (DSPE-PEG2000) and 40 parts of soybean lecithin were dissolved in a mixture of chloroform and methanol (volume ratio of 2:1), ultrasonically mixed evenly, and then the solvent was removed by rotary evaporation. The mixture was vacuum-dried at 37°C to obtain a uniform lipid film. An appropriate amount of phosphate buffer was added, and the mixture was heated and stirred at 65°C for 20 minutes to fully hydrate it. The hydrated solution was ultrasonically applied for 20 minutes, and then passed through a 0.45μm cellulose membrane three times in an extrusion device to obtain the target drug-loaded liposome solution.

[0044] Take a small amount of liposome solution and drop it on a 200-mesh copper grid. Use an ear bulb to blow dry the solution, and then observe the microscopic morphology of the liposomes under a transmission electron microscope. The results are as follows: Figure 7 As shown in FIG. , it can be seen that the prepared liposomes are regular spherical in shape and have good dispersibility.

[0045] The unencapsulated drug was separated from the prepared liposomes by high-speed centrifugation, and the liposome encapsulation efficiency (EE%) was determined by high-performance liquid chromatography (HPLC). The EE% of the drug in the liposome refers to the ratio of the amount of drug encapsulated in the liposome to the total amount of drug administered during drug loading. The calculation results are as follows: Figure 8shown.

[0046] The control group in the figure is drug-loaded liposomes without cholesterol encapsulation, which were prepared according to the above step 3, except that the CHO-CUR complex and CHO-VES complex were replaced with equal amounts of CUR and VES.

[0047] As can be seen from the figure, the encapsulation rates of curcumin and vitamin E succinate after cholesterol modification are 95% and 93% respectively, both exceeding 90%. Compared with directly encapsulated drugs, the encapsulation rate of curcumin increased by 21.8% and the encapsulation rate of vitamin E succinate increased by 24%.

[0048] Example 2

[0049] In this example, drug-loaded liposomes were prepared using the same method as in Example 1, with the only difference being that the molar ratio of curcumin to cholesterol in step 1 was adjusted to 1:2, 1.2:2, 1.5:2, 1:2.2, and 1:2.4. Testing revealed that the encapsulation efficiencies of curcumin in the resulting drug-loaded liposomes at the aforementioned different molar ratios were 95%, 91%, 93%, 92%, and 90%, respectively. These efficiencies were significantly higher than those achieved with conventional methods, demonstrating that cholesterol-modified curcumin effectively improves curcumin encapsulation efficiency. Furthermore, the experimental results showed that the optimal encapsulation effect occurred at a curcumin to cholesterol molar ratio of 1:2, achieving the highest encapsulation efficiency of 95%. In contrast, the encapsulation efficiency at a molar ratio of 1:2.4, while still high (90%), was slightly lower than that achieved with the other ratio combinations.

[0050] This series of experimental results demonstrates that the molar ratio of curcumin to cholesterol significantly influences the encapsulation efficiency of drug-loaded liposomes. A suitable curcumin to cholesterol ratio can improve encapsulation efficiency. This result provides valuable insights for further optimizing liposome preparation processes and suggests that in practical applications, the appropriate ratio should be selected based on specific needs to achieve optimal encapsulation.

[0051] Example 3

[0052] In this example, drug-loaded liposomes were prepared using the same method as in Example 1, with the only difference being that the molar ratio of vitamin E succinate to cholesterol in step 2 was adjusted to 1:1, 1.1:1, 1.2:1, 1:1.1, and 1:1.2. Testing revealed that the encapsulation efficiency of vitamin E succinate in the drug-loaded liposomes at these different molar ratios was 90%, 92%, 93%, 89%, and 88%, respectively. Compared with traditional preparation methods, the use of cholesterol-modified vitamin E succinate significantly improved the encapsulation efficiency of the liposomes, demonstrating superior encapsulation performance. Further analysis revealed that the optimal encapsulation effect occurred at a vitamin E succinate to cholesterol molar ratio of 1.2:1, reaching a maximum encapsulation efficiency of 93%. While the encapsulation efficiency at a molar ratio of 1:1.2 remained high (88%), it was slightly lower than at the other ratio combinations.

[0053] This series of experimental results demonstrates that the molar ratio of vitamin E succinate to cholesterol significantly influences the encapsulation efficiency of drug-loaded liposomes. A suitable ratio not only improves encapsulation efficiency but also significantly enhances liposome performance. Notably, when the ratio deviates from the optimal value (e.g., 1:1.2), the encapsulation efficiency decreases slightly. Therefore, to achieve optimal encapsulation results in practical applications, the optimal molar ratio of vitamin E succinate to cholesterol should be precisely selected based on specific needs. This study provides a useful reference for further optimizing liposome preparation processes.

[0054] Example 4

[0055] In this example, drug-loaded liposomes were prepared according to the same method as in Example 1, with the only difference being that the hydration temperature in step 3 was adjusted to 55°C, 60°C, 65°C, 70°C, and 75°C. After testing, the encapsulation efficiency of curcumin showed certain fluctuations at the aforementioned different reaction temperatures. As the temperature increased, the encapsulation efficiency gradually increased until it reached 95% and 94% at 65°C and 70°C, respectively, reaching a maximum value. Higher temperatures may help improve the stability of the liposome membrane and promote uniform encapsulation of curcumin. However, when the temperature was further increased to 75°C, the encapsulation efficiency decreased slightly to 90%. This phenomenon may be due to the high temperature causing excessive fluidity of the liposome membrane, affecting the stability of the liposomes, thereby slightly reducing the encapsulation effect of curcumin.

[0056] This series of experimental results shows that temperature has a significant impact on the preparation process of drug-loaded liposomes. Higher temperatures facilitate the formation of the liposome membrane and better encapsulation of curcumin, while lower temperatures may lead to incomplete liposome formation or low curcumin encapsulation efficiency. Therefore, reasonable control of reaction temperature is a key factor in improving the encapsulation efficiency of curcumin. Based on the experimental results, a reaction temperature of 65°C is the optimal temperature condition in this method, which helps to obtain drug-loaded liposomes with a high encapsulation efficiency.

[0057] The above are merely exemplary embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for improving liposome encapsulation efficiency by modifying cholesterol composite prodrugs, characterized in that: First, the drugs curcumin and vitamin E succinate are reacted with cholesterol separately to obtain cholesterol-curcumin complex and cholesterol-vitamin E succinate complex. The two complexes are then uniformly mixed with lipids and subjected to thin film hydration to prepare drug-loaded liposomes. The cholesterol-curcumin complex is prepared by dissolving curcumin and cholesterol in acetone at a molar ratio of 1:2, stirring and reacting at 40-60° C. for 1-3 hours, and then removing the acetone by rotary evaporation and vacuum drying to obtain the cholesterol-curcumin complex. The preparation method of the cholesterol-vitamin E succinate complex comprises: adding vitamin E succinate to an organic solvent, then adding a catalyst and cholesterol, stirring and reacting at room temperature under nitrogen protection, washing and rotary evaporation to obtain a crude product after the reaction, and then purifying the crude product to obtain the cholesterol-vitamin E succinate complex; the molar ratio of vitamin E succinate to cholesterol is 1.2:1; The steps of preparing drug-loaded liposomes by thin film hydration method are as follows: dissolving 1-10 parts of cholesterol-curcumin complex, 1-10 parts of cholesterol-vitamin E succinate complex, 1-20 parts of cholesterol, 1-20 parts of polyethylene glycol-distearoyl phosphatidylethanolamine and 10-100 parts of soybean lecithin in a mixture of chloroform and methanol, mixing them uniformly by ultrasonication, then removing the solvent by rotary evaporation to obtain a uniform lipid film, adding phosphate buffer, heating and stirring at 60-65°C for 10-30 minutes to fully hydrate the solution; ultrasonicating the hydrated solution for 10-30 minutes, and then filtering it through a 0.45μm cellulose membrane to obtain the target drug-loaded liposome solution.

2. The method for improving liposome encapsulation efficiency by modifying cholesterol complex prodrug according to claim 1, characterized in that: The catalysts used are 4-dimethylaminopyridine DMAP and 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC, and the mass of DMAP accounts for 10-15% of the total mass of vitamin E succinate and cholesterol, and the mass of EDC accounts for 15-20% of the total mass of vitamin E succinate and cholesterol.

3. A drug-loaded liposome with high encapsulation efficiency prepared by the method according to any one of claims 1 to 2.

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