Method for improving liposome encapsulation efficiency through cholesterol composite prodrug modification

Abstract

The invention discloses a method for improving liposome encapsulation efficiency through cholesterol composite prodrug modification, which specifically comprises the following steps: firstly, respectively reacting curcumin and vitamin E succinate with cholesterol to obtain a cholesterol-curcumin compound and a cholesterol-vitamin E succinate compound; and uniformly mixing the two obtained compounds with lipid, and then preparing the drug-loaded liposome through a film hydration method. According to the invention, the problem of low solubility of curcumin and vitamin E succinate in the liposome is improved by using a method of compounding cholesterol with the prodrug, the encapsulation efficiency of the curcumin and vitamin E succinate in the liposome is improved, and a new thought is provided for preparation of poorly soluble compound liposome.

Description

Technical Field

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

[0002] Improving the in vivo bioavailability of poorly soluble compounds has always been an important topic in pharmaceutical research. However, many poorly soluble compounds are not only difficult to dissolve in water, but also lipid-water insoluble, that is, they are also difficult to dissolve in commonly used solvents under physiological conditions, which poses a great challenge to the design and application of drug dosage forms.

[0003] As a drug delivery system, liposomes can enter tumor tissues through the gaps between capillary epithelial cells when the drugs are delivered to tumor blood vessels, thereby achieving a certain tumor targeting effect. The passive targeting characteristics of the liposome drug delivery system can change the distribution of drugs in the body, which can not only improve the efficacy but also reduce the toxicity of drugs. It is an ideal carrier for increasing the drug loading of poorly soluble compounds. Through the application of liposomes, the problem of low solubility of poorly soluble drugs is solved, the toxic side effects are reduced, and at the same time, it has better targeted distribution characteristics.

[0004] Although liposomes can effectively improve the solubility of poorly soluble compounds, the encapsulation rate of most liposome drug delivery systems still cannot reach the 85% standard for injection marketing. In order to further improve the encapsulation rate, researchers have proposed some improvement plans, including: improving the preparation method (such as preparing precursor liposomes); adding surfactants to the prescription to prepare vesicles; adding surfactants and skeleton materials to prepare liposome solid nanoparticles. However, these improvement plans still have certain limitations, especially for some lipid-insoluble compounds, the encapsulation rate is still difficult to significantly improve, and the addition of surfactants to the prescription may cause strong toxic side effects. Therefore, how to reduce possible toxic side effects while improving the liposome encapsulation rate is still one of the difficulties in research. Summary of the invention

[0005] Based on the above problems in the prior art, the present invention provides a method for improving the liposome encapsulation efficiency by modifying the cholesterol composite 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 scheme: A method for improving liposome encapsulation efficiency by cholesterol composite prodrug modification, characterized in that: firstly, curcumin and vitamin E succinate are reacted with cholesterol to obtain cholesterol-curcumin complex and cholesterol-vitamin E succinate complex, and then the obtained two complexes are uniformly mixed with lipids and then prepared by thin film hydration method to obtain drug-loaded liposomes. Specifically, the steps include: Step 1: Preparation of cholesterol-curcumin complex (CHO-CUR) by solvent evaporation method Curcumin (CUR) and cholesterol (CHO) were dissolved in acetone, stirred for reaction, and then the acetone was removed by rotary evaporation and vacuum drying to obtain cholesterol-curcumin complex (CHO-CUR).

[0007] 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.

[0008] Step 2: Synthesis of cholesterol-vitamin E succinate complex (CHO-VES) by esterification reaction Vitamin E succinate (VES) is added to an organic solvent, and then a catalyst and cholesterol are added, and the mixture is stirred at room temperature under nitrogen protection. After the reaction is completed, a crude product is obtained by washing and rotary evaporation, and then purified to obtain a cholesterol-vitamin E succinate complex (CHO-VES).

[0009] 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.

[0010] Preferably, the organic solvent is anhydrous dichloromethane.

[0011] Preferably, the washing is performed sequentially with a 5% citric acid aqueous solution and a saturated saline solution.

[0012] Preferably, the purification is carried out by column chromatography using petroleum ether and ethyl acetate.

[0013] Step 3: Prepare drug-loaded liposomes by thin film hydration method 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 are dissolved in a mixture of chloroform and methanol, and ultrasonically mixed to be uniform, and then the solvent is removed by rotary evaporation to obtain a uniform lipid film, and a phosphate buffer solution is added, and heated and stirred to fully hydrate it; the hydrated solution is ultrasonically and filtered to obtain the target drug-loaded liposome solution.

[0014] 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.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention utilizes cholesterol to react with curcumin (CUR) or vitamin E succinate (VES), and can improve the solubility of the two drugs in the liposome in the subsequent liposome preparation process, thereby greatly improving the encapsulation rate and stability of the two drugs in the liposome.

[0016] The method of the invention does not use a surfactant, thereby reducing the toxic and side effects of drugs.

[0017] 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.

[0018] The method of the present invention is applicable to poorly soluble compounds, can significantly improve the encapsulation rate of poorly soluble compounds (especially lipid-water insoluble compounds), and provides a new idea for the preparation of poorly soluble compound liposomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the synthesis of CHO-CUR; Figure 2 For cholesterol 1 H NMR spectra; Figure 3 For curcumin 1 H NMR spectra; Figure 4 is the CHO-CUR prepared in the example 1 H NMR spectra; Figure 5 Schematic diagram of the synthesis of CHO-VES; Figure 6 is the CHO-VES prepared in the example 1 H NMR spectra; Figure 7 TEM image of the drug-loaded liposome prepared in the example; Figure 8 This is a graph showing the encapsulation efficiency of the drug-loaded liposomes prepared in the examples. DETAILED DESCRIPTION

[0020] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0021] Example 1 In this example, drug-loaded liposomes were prepared according to the following steps: Step 1: Prepare CHO-CUR 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.

[0022] Cholesterol, curcumin and CHO-CUR 1 H NMR test, the 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, which comes 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, which comes from cholesterol, indicating that the CHO-CUR complex was successfully synthesized.

[0023] Step 2: Preparation of CHO-VES 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 (at this time the molar ratio of VES to CHO is 1.2:1) in sequence, and stir the reaction at room temperature for 12 hours under nitrogen protection. 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 the 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.

[0024] 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, which comes from vitamin E succinate, and the peaks at 1.65 and 1.9 ppm correspond to hydrogen on the five-membered ring, which comes 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.

[0025] Step 3: Preparation of drug-loaded liposomes 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, and vacuum dried at 37°C to obtain a uniform lipid film, and an appropriate amount of phosphate buffer was added, heated and stirred at 65°C for 20 minutes to fully hydrate it; the hydrated solution was ultrasonically treated for 20 minutes, and then passed through a 0.45μm cellulose membrane in an extrusion device for 3 times to obtain the target drug-loaded liposome solution.

[0026] Take a small amount of liposome solution and drop it on a 200-mesh copper grid. Use an ear-cleaning 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, it can be seen that the prepared liposomes are regular spherical in shape and have good dispersibility.

[0027] 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 8 shown.

[0028] The control group in the figure is drug-loaded liposomes without cholesterol encapsulation, and the preparation method thereof is carried out according to the above step 3, except that the CHO-CUR complex and the CHO-VES complex are replaced with equal amounts of CUR and VES.

[0029] 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%.

[0030] Example 2 In this embodiment, drug-loaded liposomes are prepared in the same manner as in Example 1, except that the molar ratio of curcumin to cholesterol in step 1 is adjusted to 1:2, 1.2:2, 1.5:2, 1:2.2, and 1:2.4. After testing, under the aforementioned different molar ratio combinations, the encapsulation efficiency of curcumin in the prepared drug-loaded liposomes is 95%, 91%, 93%, 92%, and 90%, respectively, which are significantly higher than the encapsulation efficiency of the drug-loaded liposomes obtained by the traditional method, indicating that the encapsulation efficiency of curcumin can be effectively improved by modifying curcumin with cholesterol. In addition, the experimental results also show that the best encapsulation effect occurs when the molar ratio of curcumin to cholesterol is 1:2, and the encapsulation efficiency is the highest, reaching 95%. In contrast, when the molar ratio is 1:2.4, although the encapsulation efficiency is still high (90%), it is slightly lower than other ratio combinations.

[0031] This series of experimental results show that the molar ratio of curcumin to cholesterol has an important influence on the encapsulation efficiency of drug-loaded liposomes. The appropriate ratio of curcumin to cholesterol can improve the encapsulation efficiency. This result provides a valuable reference for further optimizing the preparation process of liposomes, and suggests that in practical applications, the appropriate ratio should be selected according to specific needs to achieve the best encapsulation effect.

[0032] Example 3 In this example, drug-loaded liposomes were prepared in the same manner as in Example 1, except 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. After testing, the encapsulation efficiency of vitamin E succinate in drug-loaded liposomes was 90%, 92%, 93%, 89%, and 88% under the aforementioned different molar ratio combinations. Compared with the traditional preparation method, the use of cholesterol-modified vitamin E succinate significantly improved the encapsulation efficiency of liposomes and showed better encapsulation performance. Further analysis found that the best encapsulation effect occurred when the molar ratio of vitamin E succinate to cholesterol was 1.2:1, and the encapsulation efficiency at this time reached a maximum value of 93%. When the molar ratio was 1:1.2, although the encapsulation efficiency was still high (88%), it was slightly lower than other ratio combinations.

[0033] This series of experimental results show that the molar ratio of vitamin E succinate to cholesterol has a significant effect on the encapsulation efficiency of drug-loaded liposomes. The appropriate ratio can not only improve the encapsulation efficiency, but also the optimized ratio can significantly improve the performance of the liposomes. It is worth noting that when the ratio deviates from the optimal value (such as 1:1.2), the encapsulation efficiency will decrease slightly. Therefore, in order to obtain the best encapsulation effect in practical applications, the optimal molar ratio of vitamin E succinate to cholesterol should be accurately selected according to specific needs. This study provides a useful reference for further optimizing the liposome preparation process.

[0034] Example 4

[0035] In this embodiment, drug-loaded liposomes were prepared in the same manner 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 the encapsulation efficiency 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 excessive fluidity of the liposome membrane caused by high temperature, which affected the stability of the liposome, thereby slightly reducing the encapsulation effect of curcumin.

[0036] This series of experimental results show that temperature has a significant effect on the preparation process of drug-loaded liposomes. Higher temperatures are conducive to the formation of liposome membranes and better encapsulation of curcumin, while lower temperatures may lead to incomplete liposome formation or low encapsulation efficiency of curcumin. 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.

[0037] The above are merely exemplary embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for improving liposome encapsulation efficiency by modifying cholesterol composite prodrugs, characterized in that: First, the drug curcumin and vitamin E succinate are reacted with cholesterol separately to obtain a cholesterol-curcumin complex and a cholesterol-vitamin E succinate complex. Then, the two complexes are uniformly mixed with lipids and then the drug-loaded liposomes are prepared by a thin film hydration method.

2. The method for improving liposome encapsulation efficiency by modifying cholesterol complex prodrug according to claim 1, characterized in that: The preparation method of the cholesterol-curcumin complex comprises: dissolving curcumin and cholesterol in acetone, stirring for reaction, and then removing the acetone by rotary evaporation and vacuum drying to obtain the cholesterol-curcumin complex.

3. The method for improving liposome encapsulation efficiency by modifying cholesterol composite prodrug according to claim 2, characterized in that: 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.

4. The method for improving liposome encapsulation efficiency by cholesterol complex prodrug modification according to claim 1, characterized in that: 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, and after the reaction is completed, washing and rotary evaporation to obtain a crude product, and then purifying to obtain the cholesterol-vitamin E succinate complex.

5. The method for improving liposome encapsulation efficiency by cholesterol complex prodrug modification according to claim 4, characterized in that: The molar ratio of vitamin E succinate and cholesterol is 1.2:1, and the catalysts used are 4-dimethylaminopyridine DMAP and 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC. 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.

6. The method for improving liposome encapsulation efficiency by cholesterol complex prodrug modification according to claim 1, characterized in that: 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 to fully hydrate it; and subjecting the hydrated solution to ultrasonication and filtration to obtain the target drug-loaded liposome solution.

7. The method for improving liposome encapsulation efficiency by cholesterol complex prodrug modification according to claim 6, characterized in that: 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.

8. A drug-loaded liposome with high encapsulation efficiency prepared by the method according to any one of claims 1 to 7.

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