Polyethylene glycol-modified liposomes loaded with dimethylcurcumin and their preparation method

Abstract

This invention relates to the field of biomedical technology, specifically to a polyethylene glycol-modified liposome loaded with dimethylcurcumin and its preparation method. First, cholesterol is reacted with succinic anhydride to obtain cholesterol monosuccinate. Then, the cholesterol monosuccinate is coupled with polyethylene glycol via an ester bond to synthesize polyethylene glycol-cholesterol. Using polyethylene glycol-cholesterol, polyethylene glycol monostearate, and lecithin as the main components, and dimethylcurcumin as the model drug, polyethylene glycol-modified dimethylcurcumin liposomes are prepared using a thin-film hydration method. These liposomes exhibit high encapsulation efficiency. In vitro simulated drug release experiments show that these liposomes can improve the sustained-release effect of dimethylcurcumin. Cytotoxicity experiments also demonstrate that the polyethylene glycol-modified dimethylcurcumin liposomes possess good antitumor activity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a polyethylene glycol-modified liposome loaded with dimethylcurcumin and its preparation method. Background Technology

[0002] Liposome technology is a technique for encapsulating active ingredients or drugs within tiny, uniquely structured vesicles composed of phospholipids (such as lecithin). This technology was first applied in the pharmaceutical industry and has gradually become one of the earliest and most mature types of novel targeted formulations used in clinical practice. As drug carriers, liposomes significantly enhance therapeutic efficacy and reduce toxicity by improving drug targeting and bioavailability.

[0003] Polyethylene glycol (PEG) liposomes represent an important development direction in liposome technology. PEGylated liposomes form a coating layer on the surface of lipid particles by covalently binding polyethylene glycol (PEG) to the lipid material, thereby increasing the stability and biocompatibility of drug particles. PEGylated liposomes offer significant advantages such as prolonged blood circulation time, increased drug accumulation at disease sites, reduced toxicity, and reduced dosing frequency.

[0004] Currently, polyethylene glycol-modified liposomes are mainly prepared using polyethylene glycol-modified lecithin and various other excipients. This preparation method is complex, costly, and results in unsatisfactory encapsulation rates. Therefore, developing a simple, efficient, and low-cost method for preparing polyethylene glycol-modified liposomes is a crucial technical challenge that urgently needs to be addressed. Summary of the Invention

[0005] The first objective of this invention is to provide a novel polyethylene glycol-modified liposome that does not require the use of polyethylene glycol-modified lecithin, has low preparation cost, high encapsulation efficiency, and good stability.

[0006] A second objective of this invention is to provide a method for preparing polyethylene glycol-modified liposomes, comprising the following steps:

[0007] (1) Preparation of succinic acid monocholesterol ester

[0008] The reaction formula for the preparation of succinic acid monocholesterol ester is as follows:

[0009]

[0010] Succinic anhydride, cholesterol, and 4-dimethylaminopyridine (DMAP) were dissolved in dichloromethane (DCM), heated to 40°C, and stirred for 24 hours. After the reaction was completed, 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] 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] Succinate monocholesterol ester, polyethylene glycol-2000 (PEG-2000), 4-dimethylaminopyridine (DMAP), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) were dissolved in dichloromethane (DCM), heated to 40°C, and stirred for 24 hours. After the reaction was completed, the solvent was removed by rotary evaporation, and the mixture was recrystallized from isopropanol and dried to obtain a white solid polyethylene glycol-cholesterol.

[0016] The molar ratio of succinic acid monocholesterol ester, polyethylene glycol 2000, EDCI and DMAP is 1:1:10:0.5.

[0017] (3) Preparation of polyethylene glycol-modified liposomes

[0018] Polyethylene glycol-cholesterol, polyethylene glycol (n=40) monostearate, lecithin and the drug dimethylcurcumin were dissolved in an organic solvent. After removing the organic solvent by rotary evaporation, ultrapure water was added, and the mixture was sonicated for 10 minutes. The supernatant was then collected by centrifugation to obtain the drug-loaded liposome solution.

[0019] The mass ratio of polyethylene glycol-cholesterol, polyethylene glycol monostearate, lecithin, and the 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 centrifugation speed is 4000 rpm; and the centrifugation time is 10 minutes.

[0020] The advantages of this invention are:

[0021] This invention uses polyethylene glycol monostearate and polyethylene glycol-cholesterol to prepare polyethylene glycol-modified liposomes, which greatly reduces the preparation cost. Furthermore, the polyethylene glycol-modified liposomes prepared by this invention have simple components, a simple preparation method, and the prepared liposomes have high encapsulation efficiency and good stability. Attached Figure Description

[0022] Figure 1 The image shows the 1H NMR spectrum of the succinic acid monocholesterol ester prepared in Example 1 of this invention.

[0023] Figure 2 The image shows the hydrogen nuclear magnetic resonance spectrum of polyethylene glycol-cholesterol prepared in Example 2 of this invention.

[0024] Figure 3 The infrared spectrum of polyethylene glycol-cholesterol prepared in Example 2 of this invention.

[0025] Figure 4 The particle size distribution of polyethylene glycolated dimethylcurcumin liposomes prepared in Example 3 of this invention is shown in the diagram. Detailed Implementation

[0026] The present invention will now be described in detail with reference to specific embodiments.

[0027] Example 1: Preparation of succinic acid monocholesterol ester

[0028] Add 10 mL of dichloromethane, 2.0 g of cholesterol, 517 mg of succinic anhydride, and 315 mg of 4-dimethylaminopyridine to a 50 mL round-bottom flask. Stir to dissolve, heat to 40 °C and stir to react for 24 hours. After the reaction is complete, remove the solvent by rotary evaporation, wash twice with dilute hydrochloric acid and 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 using proton nuclear magnetic resonance spectroscopy. Figure 1 As shown, the 1H NMR spectrum showed characteristic peaks of methylene hydrogen in succinic anhydride at chemical shifts of 2.61 and 2.67 ppm, and characteristic peaks of cholesterol 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 succinate monocholesterol ester, 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 for 24 hours. After the reaction is complete, remove the solvent by rotary evaporation, recrystallize from isopropanol, and dry to obtain a white solid polyethylene glycol-cholesterol (yield 75.3%).

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

[0033] Example 3: Preparation of polyethylene glycol-modified liposomes

[0034] Add 50 mL of anhydrous ethanol to a 100 mL round-bottom flask, then add 1 g of polyethylene glycol-cholesterol, 1 g of polyethylene glycol (n=40) monostearate, 3 g of lecithin, and 100 mg of dimethyl curcumin. Stir to dissolve, remove the solvent by rotary evaporation and form a film. Add 50 mL of ultrapure water, sonicate for 10 minutes (90 W), and centrifuge at 4000 rpm to collect the supernatant. The encapsulation efficiency of dimethyl curcumin was determined to be 94% by high-performance liquid chromatography (HPLC). The encapsulation efficiency is calculated using the following formula:

[0035]

[0036] Example 4: Preparation of polyethylene glycol-modified liposomes

[0037] Add 50 mL of anhydrous ethanol to a 100 mL round-bottom flask, then add 2 g of polyethylene glycol-cholesterol, 2 g of polyethylene glycol (n=40) monostearate, 6 g of lecithin, and 100 mg of dimethyl curcumin. Stir to dissolve, remove the solvent by rotary evaporation to form a film, add 50 mL of ultrapure water, sonicate for 10 minutes (90 W), and centrifuge at 4000 rpm to collect the supernatant. The encapsulation efficiency of dimethyl curcumin was determined to be 98% by high-performance liquid chromatography.

[0038] Example 5: Preparation of polyethylene glycol-modified liposomes

[0039] Add 50 mL of dichloromethane to a 100 mL round-bottom flask, then add 2 g of polyethylene glycol-cholesterol, 2 g of polyethylene glycol (n=40) monostearate, 6 g of lecithin, and 100 mg of dimethylcurcumin. Stir to dissolve, remove the solvent by rotary evaporation to form a film, add 50 mL of ultrapure water, sonicate for 10 minutes (90 W), and centrifuge at 4000 rpm to collect the supernatant. The encapsulation efficiency of dimethylcurcumin was determined to be 93% by high-performance liquid chromatography.

[0040] Example 6: Preparation of polyethylene glycol-modified liposomes

[0041] Add 50 mL of anhydrous ethanol to a 100 mL round-bottom flask, then add 1.5 g of polyethylene glycol-cholesterol, 0.5 g of polyethylene glycol (n=40) monostearate, 3 g of lecithin, and 100 mg of dimethylcurcumin. Stir to dissolve, remove the solvent by rotary evaporation to form a film, add 50 mL of ultrapure water, sonicate for 10 minutes (90 W), and centrifuge at 4000 rpm to collect the supernatant. The encapsulation efficiency of dimethylcurcumin was determined to be 85% by high-performance liquid chromatography.

[0042] Example 7: Preparation of polyethylene glycol-modified liposomes

[0043] Add 50 mL of anhydrous ethanol to a 100 mL round-bottom flask, then add 0.5 g of polyethylene glycol-cholesterol, 1.5 g of polyethylene glycol (n=40) monostearate, 3 g of lecithin, and 100 mg of dimethyl curcumin. Stir to dissolve, remove the solvent by rotary evaporation to form a film, add 50 mL of ultrapure water, sonicate for 10 minutes (90 W), and centrifuge at 4000 rpm to collect the supernatant. The encapsulation efficiency of dimethyl curcumin was determined to be 88% by high-performance liquid chromatography.

[0044] Comparative Example 1: Preparation of Polyethylene Glycol-Treated Liposomes

[0045] Add 50 mL of anhydrous ethanol to a 100 mL round-bottom flask, then add 2 g of polyethylene glycol-cholesterol, 3 g of lecithin, and 100 mg of dimethylcurcumin. Stir to dissolve, remove the solvent by rotary evaporation to form a film, add 50 mL of ultrapure water, and sonicate for 10 minutes (90 W). Centrifuge at 4000 rpm and collect the supernatant. The encapsulation efficiency of dimethylcurcumin was determined to be 78% by high-performance liquid chromatography.

[0046] Comparative Example 2: Preparation of Polyethylene Glycol-Modified Liposomes

[0047] Add 50 mL of anhydrous ethanol to a 100 mL round-bottom flask, then add 2 g of polyethylene glycol (n=40) monostearate, 3 g of lecithin, and 100 mg of dimethyl curcumin. Stir to dissolve, remove the solvent by rotary evaporation to form a film, add 50 mL of ultrapure water, sonicate for 10 minutes (90 W), and centrifuge at 4000 rpm to collect the supernatant. The encapsulation efficiency of dimethyl curcumin was determined to be 81% by high-performance liquid chromatography.

[0048] Comparative Example 3: Preparation of Polyethylene Glycol-Treated Liposomes

[0049] Add 50 mL of anhydrous ethanol to a 100 mL round-bottom flask, then add 0.4 g of cholesterol, 2 g of polyethylene glycol (n=40) monostearate, 6 g of lecithin, and 100 mg of dimethyl curcumin. Stir to dissolve, remove the solvent by rotary evaporation to form a film, add 50 mL of ultrapure water, sonicate for 10 minutes (90 W), and centrifuge at 4000 rpm to collect the supernatant. The encapsulation efficiency of dimethyl curcumin was determined to be 71% by high-performance liquid chromatography.

[0050] Examples 1-7 and Comparative Examples 1-3 show that, compared with Example 3 which uses both polyethylene glycol-cholesterol and polyethylene glycol (n=40) monostearate, Comparative Example 1, which uses only polyethylene glycol-cholesterol, results in a lower encapsulation efficiency, and Comparative Example 2, which uses only polyethylene glycol (n=40) monostearate, also results in a lower encapsulation efficiency. This indicates that polyethylene glycol-cholesterol and polyethylene glycol (n=40) monostearate can work synergistically in the preparation of liposomes to improve drug loading efficiency. They can interact with lecithin through their hydrophobic cholesterol and fatty acid chains. Drug loading function; compared with Example 3, the amount of polyethylene glycol-cholesterol, polyethylene glycol (n=40) monostearate, and lecithin in Example 4 was doubled, and the encapsulation efficiency was slightly improved; compared with Example 4, dichloromethane was used instead of anhydrous ethanol as a solvent in Example 5, and the encapsulation efficiency was slightly reduced; compared with Example 3, the ratio of polyethylene glycol-cholesterol and polyethylene glycol (n=40) monostearate changed in Examples 6 and 7, 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] Experimental Example 1: Determination of Particle Size and Potential

[0052] Preparation of test solution: Dilute the liposomes prepared in Examples 1-7 and Comparative Examples 1-3 with ultrapure water by 10 times to obtain the test solution.

[0053] Particle size and potential determination: Take 1 ml of each of the above-prepared test solutions and determine the particle size and potential using a Malvern particle size analyzer.

[0054] Figure 4 This is a particle size distribution diagram of the polyethylene glycol-modified liposomes prepared in Example 3 of the present invention. Experimental results show that the liposomes prepared in Example 3 have smaller particle sizes compared to Comparative Examples 1-3, which is beneficial for long-term blood circulation and tumor targeting. The liposomes prepared in Example 4 have particle sizes comparable to those in Example 3. The liposomes prepared in Example 5 have slightly larger particle sizes than those in Examples 3 and 4. The liposomes prepared in Examples 6 and 7 also have larger particle sizes than those in Example 3. The polydispersity index is around 0.2, indicating a narrow particle size distribution. The prepared liposomes have negative Zeta potentials, which is beneficial for long-term blood circulation.

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

[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] Experimental Example 2: Evaluation of In Vitro Simulated Drug Release

[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 the liposomes prepared in Example 3 and Comparative Example 1 of this invention was placed in a dialysis bag (molecular weight cutoff: 3500), sealed, and then immersed in 30 mL of the above-prepared release solution. The bag was placed in a shaker at 37°C and 180 rpm. 1 mL of the release solution was collected at 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, and 96 h, and 1 mL of the corresponding blank release solution was added. The release experiment was performed in triplicate for each release solution. The release experiment was conducted entirely under light-protected conditions. The cumulative release of dimethylcurcumin was calculated by measuring the absorbance of the collected release solution.

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

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

[0064]

[0065] Table 2 shows that Example 3 exhibits similar drug release behavior in release media at pH 7.4 and pH 5.0, with 19.6% and 15.6% of dimethylcurcumin released in the first 24 hours, respectively, and cumulative release amounts of 31.3% and 22.1% at 96 hours. The release behavior of dimethylcurcumin did not change significantly under different pH conditions, indicating that Example 3 does not show a significant pH response. Comparative Example 1 showed a slow release rate and low total release amount in release media at pH 7.4 and pH 5.0, with a total release amount of only about 5%.

[0066] Experimental Example 3: In vitro antitumor test

[0067] HepG2 and 22Rv1 cells were mixed at a rate of 5 × 10⁻⁶. 3Cells were seeded at a density of 100 cells per well in 96-well plates, with each concentration performed in 5 replicates. Free dimethylcurcumin, liposomes prepared in Example 3 and Comparative Example 1 were diluted with complete culture medium to a series of concentrations (1, 2, 5, 10, 15, and 20 μg / mL) and added to 96-well plates. The plates were co-incubated with HepG2 and 22Rv1 cells for 48 h and 72 h, respectively. Then, 150 μL of CCK-8 was added to each well, and the plates were incubated at 37°C for 4 h. The absorbance at 450 nm was measured using a multi-well microplate reader, with the absorbance at 630 nm serving as a control. Cells incubated with blank culture medium were considered to have 100% viability.

[0068] In all cell lines, the tested drugs exhibited dose- and time-dependent cytotoxicity. The maximum half-maximal inhibitory concentrations (IC50) of free dimethylcurcumin, Example 3, and Comparative Example 1 against 22Rv1 and HepG2 cells were [data missing]. 50 The data are shown in Table 3. For 22Rv1 and HepG2 cells, the antitumor activity of Example 3 was stronger than that of free dimethyl curcumin and Comparative Example 1. The average IC50 values ​​of free dimethyl curcumin, Example 3, and Comparative Example 1 after incubation with 22Rv1 cells for 48 hours are shown in Table 3. 50 The values ​​were 45.2, 8.5, and 13.4 μg / mL, respectively, and the average IC50 after 72 hours of incubation was... 50 The values ​​were 23.7, 6.3, and 8.6 μg / mL, respectively. The average IC50 values ​​of free dimethylcurcumin, Example 3, and Comparative Example 1 after incubation with HepG2 for 48 hours were... 50 The values ​​were 22.9, 7.2, and 12.8 μg / mL, respectively, and the average IC50 after incubation with HepG2 for 72 hours was... 50 The values ​​were 7.6, 2.4, and 5.5 μg / mL, respectively. The results showed that, compared to free dimethylcurcumin and Comparative Example 1, the IC50 of Example 3 was significantly higher. 50 The significant reduction indicates that Example 3 significantly enhanced the in vitro antitumor effect on HepG2 and 22Rv1 cells.

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

[0070]

Claims

1. A method for preparing polyethylene glycol-modified liposomes loaded with dimethylcurcumin, characterized in that, The preparation method steps are as follows: (1) Succinic anhydride, cholesterol and 4-dimethylaminopyridine were dissolved in dichloromethane, heated and stirred to react. After the reaction was completed, the solvent was removed by rotary evaporation. The mixture was washed with dilute hydrochloric acid and ultrapure water and dried to obtain white solid succinic anhydride monocholesterol ester. (2) The succinic acid monocholesterol ester, polyethylene glycol 2000, 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride prepared in step (1) were dissolved in dichloromethane, heated and stirred to react. After the reaction was completed, the solvent was removed by rotary evaporation, and the isopropanol was recrystallized and dried to obtain a white solid, namely polyethylene glycol-cholesterol. (3) Dissolve the polyethylene glycol-cholesterol, polyethylene glycol monostearate, lecithin and dimethylcurcumin prepared in step (2) in an organic solvent, remove the organic solvent by rotary evaporation, add ultrapure water, sonicate, centrifuge and take the supernatant to obtain the 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℃, 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 succinic acid monocholesterol ester, polyethylene glycol 2000, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine is 1:1:10:0.5, the heating and stirring reaction temperature is 40℃, 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; 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 90W and the ultrasonic time is 10 minutes; the centrifugation speed is 4000 rpm and the centrifugation time is 10 minutes.

6. A polyethylene glycol-modified liposome loaded with dimethylcurcumin prepared by the method according to any one of claims 1-5.

7. The polyethylene glycol-modified liposome loaded with dimethylcurcumin according to claim 6, characterized in that, The encapsulation efficiency of the polyethylene glycol-modified liposomes loaded with dimethylcurcumin was 85-98%.

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