Polyethylene glycol liposome loaded with dimethyl curcumin and preparation method thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to a dimethyl curcumin-loaded pegylated liposome and a preparation method thereof. The preparation method comprises the following steps: reacting cholesterol with succinic anhydride to obtain monocholesteryl succinate, and coupling the monocholesteryl succinate with polyethylene glycol through an ester bond to synthesize polyethylene glycol-cholesterol. Polyethylene glycol-cholesterol, polyethylene glycol monostearate and lecithin are used as main components, dimethyl curcumin is used as a model drug, and the pegylated dimethyl curcumin liposome is prepared by a film hydration method. The liposome has high encapsulation efficiency. An in-vitro simulated drug release experiment shows that the liposome can improve the slow release effect of the dimethyl curcumin. A cytotoxicity experiment also shows that the pegylated dimethyl curcumin liposome has relatively good anti-tumor activity.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a PEGylated liposome carrying dimethylcurcumin and a preparation method thereof. Background Art

[0002] Liposome technology is a technology that encapsulates active ingredients or drugs in tiny, uniquely structured vesicles composed of phospholipids (such as lecithin). This technology was first used in the pharmaceutical industry and has gradually become the earliest and most mature type of new targeted preparations in clinical application. As a drug carrier, liposomes significantly improve the therapeutic effect of drugs and reduce toxicity by improving the targeting and bioavailability of drugs.

[0003] PEGylated liposomes are an important development direction of liposome technology. PEGylated liposomes form a covering layer on the surface of lipid particles by covalently bonding polyethylene glycol (PEG) to lipid materials, thereby increasing the stability and biocompatibility of drug particles. PEGylated liposomes have significant advantages such as prolonging blood circulation time, increasing drug accumulation at the disease site, reducing toxicity and administration frequency.

[0004] At present, PEGylated liposomes are mainly prepared by using PEGylated lecithin and a variety of other excipients. The preparation method is relatively complicated, costly, and the encapsulation rate is not ideal. Therefore, the development of a simple, efficient, and low-cost new method for preparing PEGylated liposomes is a key technical problem that needs to be solved urgently. Summary of the invention

[0005] The first object of the present invention is to provide a novel PEGylated liposome, which does not require the use of PEGylated lecithin, has low preparation cost, high encapsulation efficiency and good stability.

[0006] The second object of the present invention is to provide a method for preparing PEGylated liposomes, which is carried out according to the following steps:

[0007] (1) Preparation of monocholesterol succinate

[0008] The preparation reaction formula of monocholesterol succinate is as follows:

[0009]

[0010] Succinic anhydride, cholesterol and 4-dimethylaminopyridine (DMAP) were dissolved in dichloromethane (DCM), heated to 40°C and stirred for reaction for 24 hours. After the reaction, the solvent was removed by rotary evaporation, and the mixture was washed with dilute hydrochloric acid and ultrapure water respectively, and dried to obtain a white solid.

[0011] Wherein, the molar ratio of succinic anhydride, cholesterol and 4-dimethylaminopyridine is 1:1:0.5.

[0012] (2) Preparation of polyethylene glycol-cholesterol

[0013] The reaction formula for preparing polyethylene glycol-cholesterol is as follows:

[0014]

[0015] Dissolve monocholesterol succinate, polyethylene glycol-2000 (PEG-2000), 4-dimethylaminopyridine (DMAP) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) in dichloromethane (DCM), heat to 40°C and stir to react for 24 hours. After the reaction, remove the solvent by rotary evaporation, recrystallize with isopropanol, and dry to obtain white solid polyethylene glycol-cholesterol.

[0016] Wherein, the molar ratio of monocholesterol succinate, polyethylene glycol 2000, EDCI and DMAP is 1:1:10:0.5.

[0017] (3) Preparation of PEGylated liposomes

[0018] Polyethylene glycol-cholesterol, polyethylene glycol (n=40) monostearate, lecithin and drug dimethyl curcumin were dissolved in an organic solvent, the organic solvent was removed by rotary evaporation, ultrapure water was added, ultrasonication was performed for 10 minutes, and the supernatant was collected by centrifugation to obtain a drug-loaded liposome solution.

[0019] Among them, the mass ratio of polyethylene glycol-cholesterol, polyethylene glycol monostearate, lecithin and drug is 0.5-2:0.5-2:3-6:0.1; the organic solvent is anhydrous ethanol or dichloromethane; the ultrasonic power is 90W; the centrifugal speed is 4000rpm, and the centrifugal time is 10 minutes.

[0020] The advantages of the present invention are:

[0021] The invention uses polyethylene glycol monostearate and polyethylene glycol-cholesterol to prepare PEGylated liposomes, so the preparation cost is greatly reduced. The invention also has simple ingredients and a simple preparation method, and the prepared liposomes have high encapsulation efficiency and good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of monocholesterol succinate prepared in Example 1 of the present invention.

[0023] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of polyethylene glycol-cholesterol prepared in Example 2 of the present invention.

[0024] Figure 3 This is the infrared spectrum of polyethylene glycol-cholesterol prepared in Example 2 of the present invention.

[0025] Figure 4 The particle size diagram of PEGylated dimethylcurcumin liposomes prepared in Example 3 of the present invention. DETAILED DESCRIPTION

[0026] The present invention is described in detail below in conjunction with specific embodiments.

[0027] Example 1 Preparation of monocholesterol succinate

[0028] In a 50-ml round-bottom flask, add 10 ml of dichloromethane, 2.0 g of cholesterol, 517 mg of succinic anhydride, and 315 mg of 4-dimethylaminopyridine, stir to dissolve, heat to 40°C and stir to react for 24 hours. After the reaction is completed, remove the solvent by rotary evaporation, wash twice with dilute hydrochloric acid, wash twice with ultrapure water, and dry to obtain a white solid (yield 87.8%).

[0029] The chemical structure of the target compound prepared in Example 1 was confirmed by hydrogen nuclear magnetic resonance spectroscopy. Figure 1 As shown, the nuclear magnetic resonance hydrogen spectrum showed characteristic peaks of methylene hydrogen in succinic anhydride at chemical shifts of 2.61 and 2.67 ppm, and a characteristic peak of cholesterol appeared at 4.65 ppm, indicating that succinic anhydride and cholesterol were successfully coupled.

[0030] Example 2 Preparation of polyethylene glycol-cholesterol

[0031] In a 50 ml round-bottom flask, add 10 ml of dichloromethane, 974 mg of monocholesterol succinate, 4 g of polyethylene glycol (molecular weight 2000), 122 mg of 4-dimethylaminopyridine, and 3.83 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, stir to dissolve, heat to 40°C and stir to react for 24 hours. After the reaction is completed, remove the solvent by rotary evaporation, recrystallize with isopropanol, and dry to obtain white solid polyethylene glycol-cholesterol (yield 75.3%).

[0032] like Figure 2 As shown, the H NMR spectrum of polyethylene glycol-cholesterol showed characteristic peaks of methylene hydrogen in succinic anhydride at chemical shifts of 2.60 and 2.65 ppm, characteristic peaks of polyethylene glycol at 3.51-3.78 ppm, and characteristic peaks of cholesterol at 4.62 ppm, indicating that the coupling of polyethylene glycol-cholesterol was successful. Figure 3 As shown, the infrared spectrum of polyethylene glycol-cholesterol copolymer showed a strong peak at 1730cm-1, which is the characteristic absorption peak of C=O in the ester bond, while PEG itself does not have this peak, which proves that the coupling of monocholesterol succinate with the terminal hydroxyl group of PEG is successful.

[0033] Example 3 Preparation of PEGylated liposomes

[0034] In a 100 ml round-bottom flask, 50 ml of anhydrous ethanol was added, followed by 1 gram of polyethylene glycol-cholesterol, 1 gram of polyethylene glycol (n=40) monostearate, 3 grams of lecithin, and 100 mg of dimethyl curcumin were stirred and dissolved, the solvent was removed by rotary evaporation and film-formed, 50 ml of ultrapure water was added, ultrasonic dispersion was performed for 10 minutes (90W), and supernatant was taken by centrifugation at 4000 rpm. The encapsulation efficiency of dimethyl curcumin measured by high performance liquid chromatography was 94%. The encapsulation efficiency calculation formula is:

[0035]

[0036] Example 4 Preparation of PEGylated liposomes

[0037] In a 100 ml round-bottom flask, 50 ml of absolute ethanol was added, followed by 2 g of polyethylene glycol-cholesterol, 2 g of polyethylene glycol (n=40) monostearate, 6 g of lecithin, and 100 mg of dimethyl curcumin were stirred and dissolved, and the solvent was removed by rotary evaporation and film-forming, 50 ml of ultrapure water was added, and ultrasonic dispersion was performed for 10 minutes (90W), and supernatant was taken by centrifugation at 4000 rpm. The encapsulation efficiency of dimethyl curcumin was measured to be 98% by high performance liquid chromatography.

[0038] Example 5 Preparation of PEGylated liposomes

[0039] In a 100 ml round-bottom flask, 50 ml of dichloromethane was added, followed by 2 g of polyethylene glycol-cholesterol, 2 g of polyethylene glycol (n=40) monostearate, 6 g of lecithin, and 100 mg of dimethyl curcumin were stirred and dissolved, and the solvent was removed by rotary evaporation and film-forming, 50 ml of ultrapure water was added, and ultrasonic dispersion was performed for 10 minutes (90W), and supernatant was taken by centrifugation at 4000 rpm. The encapsulation efficiency of dimethyl curcumin was measured by high performance liquid chromatography and was 93%.

[0040] Example 6 Preparation of PEGylated liposomes

[0041] In a 100 ml round-bottom flask, 50 ml of absolute ethanol was added, followed by 1.5 g of polyethylene glycol-cholesterol, 0.5 g of polyethylene glycol (n=40) monostearate, 3 g of lecithin, and 100 mg of dimethyl curcumin were stirred and dissolved, and the solvent was removed by rotary evaporation and film-forming, 50 ml of ultrapure water was added, and ultrasonic dispersion was performed for 10 minutes (90 W), and supernatant was taken by centrifugation at 4000 rpm. The encapsulation efficiency of dimethyl curcumin was 85% by high performance liquid chromatography.

[0042] Example 7 Preparation of PEGylated liposomes

[0043] In 100 ml round-bottom flask, add 50 ml absolute ethanol, then add 0.5 g of polyethylene glycol-cholesterol, 1.5 g of polyethylene glycol (n=40) monostearate, 3 g of lecithin, 100 mg of dimethyl curcumin stirring and dissolving, rotary evaporation to remove solvent and film forming, add 50 ml ultrapure water, ultrasonic dispersion for 10 minutes (90W), 4000rpm centrifugal supernatant. Measure the dimethyl curcumin encapsulation efficiency by high performance liquid chromatography and it is 88%.

[0044] Comparative Example 1 Preparation of PEGylated liposomes

[0045] In a 100 ml round-bottom flask, 50 ml of anhydrous ethanol was added, followed by 2 g of polyethylene glycol-cholesterol, 3 g of lecithin, and 100 mg of dimethyl curcumin, stirred and dissolved, the solvent was removed by rotary evaporation and film-formed, 50 ml of ultrapure water was added, ultrasonic dispersion was performed for 10 minutes (90 W), and the supernatant was taken by centrifugation at 4000 rpm. The encapsulation efficiency of dimethyl curcumin was measured to be 78% by high performance liquid chromatography.

[0046] Comparative Example 2 Preparation of PEGylated liposomes

[0047] In a 100 ml round-bottom flask, 50 ml of absolute ethanol was added, followed by 2 g of polyethylene glycol (n=40) monostearate, 3 g of lecithin, and 100 mg of dimethyl curcumin were stirred and dissolved, the solvent was removed by rotary evaporation and film-formed, 50 ml of ultrapure water was added, ultrasonic dispersion was performed for 10 minutes (90 W), and supernatant was taken by centrifugation at 4000 rpm. The encapsulation efficiency of dimethyl curcumin was measured to be 81% by high performance liquid chromatography.

[0048] Comparative Example 3 Preparation of PEGylated liposomes

[0049] In a 100 ml round-bottom flask, 50 ml of absolute ethanol was added, followed by 0.4 g of cholesterol, 2 g of polyethylene glycol (n=40) monostearate, 6 g of lecithin, and 100 mg of dimethyl curcumin were stirred and dissolved, and the solvent was removed by rotary evaporation and film-forming, 50 ml of ultrapure water was added, and ultrasonic dispersion was performed for 10 minutes (90 W), and supernatant was taken by centrifugation at 4000 rpm. The encapsulation efficiency of dimethyl curcumin was 71% by high performance liquid chromatography.

[0050] It can be seen from Examples 1 to 7 and Comparative Examples 1 to 3 that, compared with Example 3 using both polyethylene glycol-cholesterol and polyethylene glycol (n=40) monostearate, the use of only polyethylene glycol-cholesterol in Comparative Example 1 leads to a decrease in the encapsulation efficiency, and the use of only polyethylene glycol (n=40) monostearate in Comparative Example 2 also leads to a decrease in the encapsulation efficiency, indicating that polyethylene glycol-cholesterol and polyethylene glycol (n=40) monostearate can work synergistically in the preparation process of liposomes to improve the drug loading efficiency, and the two can be developed together with lecithin through hydrophobic cholesterol and fatty acid chains. Compared with Example 3, in Example 4, the amount of polyethylene glycol-cholesterol, polyethylene glycol (n=40) monostearate and lecithin are doubled, and the encapsulation efficiency is slightly improved; compared with Example 4, in Example 5, dichloromethane is used as the solvent instead of anhydrous ethanol, and the encapsulation efficiency is slightly reduced; compared with Example 3, the ratio of polyethylene glycol-cholesterol and polyethylene glycol (n=40) monostearate in Examples 6 and 7 is changed, resulting in a slight decrease in the encapsulation efficiency, indicating that the optimal ratio of polyethylene glycol-cholesterol and polyethylene glycol (n=40) monostearate is 1:1.

[0051] Test Example 1 Determination of Particle Size and Potential

[0052] Preparation of test solution: The liposomes prepared in Examples 1 to 7 and Comparative Examples 1 to 3 were diluted 10 times with ultrapure water to obtain a test solution.

[0053] Particle size and potential measurement: Take 1 ml of each test solution prepared above and measure the particle size and potential using a Malvern particle size analyzer.

[0054] Figure 4 The particle size distribution diagram of the PEGylated liposome prepared in Example 3 of the present invention. The experimental results show that the particle size of the liposome prepared in Example 3 is smaller than that of Comparative Examples 1 to 3, which is beneficial to long blood circulation and tumor targeting. The particle size of the liposome prepared in Example 4 is equivalent to that of Example 3. The particle size of the liposome prepared in Example 5 is slightly larger than that of Example 3 and Example 4. The particle size of the liposome prepared in Example 6 and Example 7 is also larger than that of Example 3. The polydispersity index is about 0.2, indicating a narrow particle size distribution. The Zeta potential of the prepared liposome is negative, which is beneficial to long blood circulation.

[0055] Table 1. Particle size and potential of liposomes prepared in Examples 3 to 7

[0056] sample Particle size (nm) Polydispersity index Zeta potential (mV) Example 3 77.9±8.9 0.220±0.015 -13.5±0.7 Example 4 72.3±6.5 0.234±0.015 -14.1±0.9 Example 5 86.9±8.3 0.207±0.014 -12.5±1.4 Example 6 85.3±9.5 0.216±0.033 -13.2±0.5 Example 7 90.7±5.6 0.228±0.017 -13.7±1.1 Comparative Example 1 100.6±5.3 0.252±0.023 -10.3±1.9 Comparative Example 2 115.6±5.8 0.243±0.027 -15.5±2.2 Comparative Example 3 103.4±6.6 0.217±0.018 -10.2±0.9

[0057] Test Example 2 Evaluation of simulated drug release in vitro

[0058] Based on the encapsulation efficiency and particle size test results, the liposomes prepared in Example 3 and Comparative Example 1 were selected for in vitro simulated drug release evaluation.

[0059] Preparation of pH 5.0 release solution: weigh 0.12 g potassium dihydrogen phosphate and 1.79 g disodium hydrogen phosphate dodecahydrate, add 350 ml ultrapure water, 150 ml anhydrous ethanol and 2.5 ml Tween-80 to dissolve, and then adjust the pH to 5.0.

[0060] Preparation of pH 7.4 release solution: weigh 0.12 g potassium dihydrogen phosphate and 1.79 g disodium hydrogen phosphate dodecahydrate, add 350 ml ultrapure water, 150 ml anhydrous ethanol and 2.5 ml Tween-80 to dissolve, and then adjust the pH to 7.4.

[0061] In vitro simulated drug release evaluation: 1 ml of liposomes prepared in Example 3 of the present invention and Comparative Example 1 were placed in a dialysis bag (molecular weight cutoff: 3500), sealed, and then immersed in 30 ml of the above-configured release solution, placed in a shaker for shaking, the temperature was 37 ° C, and the speed was 180 rpm. Take out 1 ml of release solution at 1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h, and 96h time points, and then add 1 ml of the corresponding blank release solution. The release experiment in each release solution was performed in parallel in three groups. The release experiment was carried out under light-proof conditions throughout the process. The cumulative release of dimethyl curcumin was calculated by detecting the absorbance of the released solution.

[0062] The cumulative release results of the liposome solutions prepared in Example 3 and Comparative Example 1 are shown in Table 2.

[0063] Table 2. Cumulative release of dimethylcurcumin liposome solution prepared in Example 3 and Comparative Example 1

[0064]

[0065] As shown in Table 2, Example 3 has similar drug release behavior in release media of pH 7.4 and pH 5.0, with 19.6% and 15.6% of dimethyl curcumin released in the first 24h, and the cumulative release amounts in 96h are 31.3% and 22.1%, respectively. The release behavior of dimethyl curcumin under different pH conditions does not change significantly, indicating that Example 3 has no obvious pH responsiveness. Comparative Example 1 has a slow release rate and a small total release amount in release media of pH 7.4 and pH 5.0, with a total release amount of only about 5%.

[0066] Test Example 3 In vitro antitumor test

[0067] HepG2 and 22Rv1 cells were cultured at 5×10 3The cells were inoculated into a 96-well plate at a density of 10 cells per well, and 5 parallel experiments were performed for each concentration. Free dimethylcurcumin, liposomes prepared in Example 3 and Comparative Example 1 were diluted with complete medium into a series of concentrations (the concentrations were 1, 2, 5, 10, 15, and 20 μg / mL, respectively) and added to a 96-well plate, and incubated with HepG2 and 22Rv1 cells for 48 h and 72 h, respectively, and then 150 μL of CCK-8 was further added to each well, incubated at 37 ° C for 4 hours, and the absorbance at 450 nm was detected in a multifunctional microplate detector. The absorbance at 630 nm was used as a control, and the cells incubated with blank medium were taken as 100% survival rate.

[0068] In all cell lines, the tested drugs showed dose- and time-dependent cytotoxicity. The maximum half inhibitory concentration (IC50) of free dimethylcurcumin, Example 3 and Comparative Example 1 on 22Rv1 and HepG2 cells, respectively, was 2.37 ± 0.13, and 1.34 ± 0.12. 50 ) data are shown in Table 3. For 22Rv1 and HepG2 cells, the antitumor activity of Example 3 is stronger than that of free dimethyl curcumin and Comparative Example 1. The average IC 50 The values ​​were 45.2, 8.5, and 13.4 μg / mL, respectively, and the average IC 50 The average IC values ​​of free dimethylcurcumin, Example 3 and Comparative Example 1 after incubation with HepG2 for 48 hours were 23.7, 6.3 and 8.6 μg / mL, respectively. 50 The values ​​were 22.9, 7.2, and 12.8 μg / mL, respectively, and the average IC 50 The values ​​were 7.6, 2.4, and 5.5 μg / mL, respectively. The results showed that compared with free dimethyl curcumin and Comparative Example 1, the IC 50 This indicates that the in vitro anti-tumor effect of Example 3 on HepG2 and 22Rv1 cells is significantly enhanced.

[0069] Table 3. In vitro antitumor activity of liposomes prepared in Example 3 and Comparative Example 1

[0070]

Claims

1. A method for preparing a PEGylated liposome loaded with dimethylcurcumin, characterized in that: The preparation method steps are as follows: (1) dissolving succinic anhydride, cholesterol and 4-dimethylaminopyridine in dichloromethane, heating and stirring to react, after the reaction is completed, removing the solvent by rotary evaporation, washing with dilute hydrochloric acid and ultrapure water respectively, and drying to obtain white solid monocholesterol succinate; (2) dissolving the monocholesterol succinate prepared in step (1), polyethylene glycol 2000, 4-dimethylaminopyridine EDCI and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride DMAP in dichloromethane, heating and stirring to react, after the reaction is completed, removing the solvent by rotary evaporation, recrystallizing with isopropanol, and drying to obtain a white solid, i.e., polyethylene glycol-cholesterol; (3) dissolving the polyethylene glycol-cholesterol, polyethylene glycol monostearate, lecithin and dimethyl curcumin prepared in step (2) in an organic solvent, removing the organic solvent by rotary evaporation, adding ultrapure water for ultrasonication, and centrifuging to obtain the supernatant to obtain a drug-loaded liposome solution.

2. The method for preparing liposomes according to claim 1, characterized in that: In step (1), the molar ratio of succinic anhydride, cholesterol and 4-dimethylaminopyridine is 1:1:0.5, the heating and stirring reaction temperature is 40° C., and the reaction time is 24 hours.

3. The method for preparing liposomes according to claim 1, characterized in that: In step (2), the molar ratio of monocholesterol succinate, polyethylene glycol 2000, EDCI and DMAP is 1:1:10:0.5, the heating and stirring reaction temperature is 40° C., and the reaction time is 24 hours.

4. The method for preparing liposomes according to claim 1, characterized in that: In step (3), the mass ratio of polyethylene glycol-cholesterol, polyethylene glycol monostearate, lecithin and dimethylcurcumin is: 0.5-2:0.5-2:3-6:0.1; and the organic solvent is anhydrous ethanol or dichloromethane.

5. The method for preparing liposomes according to claim 1, characterized in that: In step (3), the ultrasonic power is 90 W, and the ultrasonic time is 10 minutes; the centrifugal speed is 4000 rpm, and the centrifugal time is 10 minutes.

6. A PEGylated liposome loaded with dimethylcurcumin prepared according to the method according to any one of claims 1 to 5.

7. The PEGylated liposome carrying dimethylcurcumin according to claim 6, characterized in that: The encapsulation efficiency of the PEGylated liposomes carrying dimethyl curcumin is 85-98%.

8. Use of PEGylated liposomes loaded with dimethylcurcumin prepared according to the method according to any one of claims 1 to 5, characterized in that: The PEGylated liposome loaded with dimethyl curcumin is used for preparing an anti-tumor medicament.

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