Preparation method of pentagalloylglucose liposome as well as product and application of pentagalloylglucose liposome

Abstract

The invention relates to the technical field of preparation of lipidosome, in particular to a preparation method of pentagalloylglucose lipidosome as well as a product and application of the pentagalloylglucose lipidosome. The preparation method of the pentagalloylglucose liposome comprises the following steps: obtaining a lipid solution, so as to obtain a pentagalloylglucose solution; mixing and stirring the lipid solution and the pentagalloylglucose solution to obtain micelles; removing the organic solvent in the micelles to obtain a lipid membrane; mixing the lipid membrane with a surfactant and a phosphate buffer solution to obtain a mixed solution; and filtering the mixed solution to obtain the pentagalloylglucose liposome. The pentagalloylglucose liposome provided by the invention is high in encapsulation efficiency, small in particle size and good in stability.

Description

Technical Field

[0001] The invention relates to the technical field of liposome preparation, and in particular to a preparation method of pentagalloyl glucose liposome, a product thereof and an application thereof. Background Art

[0002] Pentagalloylglucose belongs to the natural polyphenol tannic acid compounds, which has multiple biological and pharmacological activities such as anti-tumor, antioxidant, anti-inflammatory, and cell protection, and has become a potential target for use in medicines and health foods. Although pentagalloylglucose is expected to be used as a chemotherapy adjuvant to reduce the dosage and toxicity of chemotherapy drugs, pentagalloylglucose is more hydrophobic than other polyphenol compounds, making it unstable in the body and having low bioavailability. Its inhibitory effect on tumor cells is time- and dose-dependent, and pentagalloylglucose is still subject to certain limitations when used directly as an adjuvant drug. Liposomes are vesicles formed by phospholipid bilayer membranes with a watery core inside. Liposomes have amphiphilic properties due to their specific structure, can efficiently load hydrophilic and hydrophobic drugs, and have good biocompatibility and tissue targeting; liposomes have been used in the field of biomedicine.

[0003] A Chinese invention patent with patent publication number CN110638820A discloses the use of a compound in the preparation of a drug for treating renal cell carcinoma. Although 1,2,3,4,6-pentagalloylglucose is used, no technical solution is provided for isolating 1,2,3,4,6-pentagalloylglucose from the external environment.

[0004] Therefore, providing a pentagalloyl glucose liposome with high encapsulation efficiency, small particle size and good stability is the main technical problem that needs to be solved at present. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a preparation method of pentagalloyl glucose liposomes, and pentagalloyl glucose is encapsulated in the vesicles of the liposomes, which can prolong the action time of the drug to a certain extent, improve the stability of pentagalloyl glucose in the body, and achieve more clear targeted controlled release and other effects. The preparation method of the pentagalloyl glucose liposomes provided by the present invention is simple, the encapsulation rate of pentagalloyl glucose is high, the obtained pentagalloyl glucose liposomes have stable properties, and the preparation cycle is short, the cost is low, and it is suitable for large-scale production.

[0006] In order to solve the above problems, the first aspect of the present invention provides a method for preparing pentagalloyl glucose liposomes, the preparation method comprising the following steps:

[0007] S1. adding liposomes into an organic solvent for dissolution to obtain a lipid solution;

[0008] S2. mixing pentagalloylglucose with a phosphate buffer solution to obtain a pentagalloylglucose solution;

[0009] S3. mixing the lipid solution and the pentagalloylglucose solution and stirring to obtain micelles;

[0010] S4. removing the organic solvent in the micelle to obtain a lipid membrane;

[0011] S5. mixing the lipid film with a surfactant and a phosphate buffer solution to obtain a mixed solution;

[0012] S6. Filter the mixed solution to obtain pentagalloylglucose liposomes.

[0013] In one embodiment, the solid-to-liquid ratio of liposomes and organic solvent in S1 is (7-9):1 mg / mL.

[0014] In one embodiment, the solid-to-liquid ratio of liposomes to organic solvent in S1 is 8:1 mg / mL.

[0015] In one embodiment, the organic solvent in S1 consists of chloroform and diethyl ether.

[0016] In one embodiment, in S1, liposomes are added to an organic solvent and stirred and dissolved for 10-30 minutes to obtain a lipid solution;

[0017] In one embodiment, the liposome in S1 is selected from a combination of one or more of cholesterol, soybean lecithin, and hydrogenated soybean lecithin.

[0018] In one embodiment, the liposome comprises soybean lecithin and cholesterol, and the mass ratio of the soybean lecithin to cholesterol is (2-4):1.

[0019] In one embodiment, the liposome is composed of soybean lecithin and cholesterol, and the mass ratio of the soybean lecithin to cholesterol is 3:1.

[0020] In one embodiment, the solid-to-liquid ratio of pentagalloylglucose to phosphate buffer solution in S2 is (1-5): 1 mg / mL.

[0021] In one embodiment, the solid-to-liquid ratio of pentagalloylglucose to phosphate buffer solution in S2 is 5:1 mg / mL.

[0022] In one embodiment, in S2, pentagalloylglucose and phosphate buffer solution are stirred and mixed for 10-30 minutes to obtain a pentagalloylglucose solution.

[0023] In one embodiment, the lipid solution and the pentagalloylglucose solution are mixed and stirred for 1.5-2 hours in S3.

[0024] In one embodiment, in S4, a rotary evaporator is used to remove the organic solvent in the micelle at 35-45° C. to obtain a lipid film.

[0025] In one embodiment, in S4, a rotary evaporator is used to remove the organic solvent in the micelle at 40° C. to obtain a lipid membrane.

[0026] In one embodiment, the surfactant in S5 includes a combination of one or more of Tween-20, Tween-40, Tween-40, and Tween-80.

[0027] In one embodiment, the surfactant in S5 is Tween-80.

[0028] In one embodiment, the pH of the phosphate buffer solution in S2 and S5 is 7.2-7.5.

[0029] In one embodiment, in S5, the lipid film obtained in S4 is mixed with a surfactant and a phosphate buffer solution, and a mixed solution is obtained by magnetic stirring.

[0030] In one embodiment, in S6, the mixed solution is allowed to stand for 1-3 hours before filtering, and a microporous filter membrane with a pore size of 0.1-0.5 μm is used to filter the particles.

[0031] In one embodiment, in S6, the mixed solution is allowed to stand for 2 hours before filtering, and a microporous filter membrane with a pore size of 0.22 μm is used to filter the particles.

[0032] In order to improve the encapsulation rate of pentagalloyl glucose liposomes, reduce their particle size, and improve their stability, the inventors found in the experimental process that the present invention uses chloroform and ether as organic solvents to dissolve soybean lecithin and cholesterol, the solid-liquid ratio of liposomes and organic solvents is (7-9): 1 mg / mL, the mass ratio of soybean lecithin and cholesterol is (2-4): 1, and the lipid solution and pentagalloyl glucose solution are mixed and stirred to obtain micelles, and the organic solvent in the micelles is removed to obtain lipid membranes; the lipid membranes are mixed with Tween -80 and phosphate buffer solution are mixed to obtain a mixed solution, the mixed solution is allowed to stand for 2 hours and then filtered, and a microporous filter membrane with a pore size of 0.22 μm is used for filtration to obtain pentagalloyl glucose liposomes with high encapsulation rate, small particle size and good stability. The possible reason is that Tween-80 is used to mix with lipid membrane and phosphate buffer solution to obtain a mixed solution, and the whole particle is filtered through a 0.22 μm microporous filter membrane, which can improve the encapsulation rate of pentagalloyl glucose and has good stability.

[0033] In one embodiment, after the pentagalloyl glucose liposomes are obtained in S6, the pentagalloyl glucose liposomes are stored at 1-8°C.

[0034] In one embodiment, after the pentagalloyl glucose liposomes are obtained in S6, the pentagalloyl glucose liposomes are stored at 4°C.

[0035] The second aspect of the present invention provides a pentagalloylglucose liposome, which is prepared by the above-mentioned method for preparing pentagalloylglucose liposome.

[0036] The third aspect of the present invention provides an application of pentagalloylglucose liposomes, wherein the pentagalloylglucose liposomes are applied in the technical field of targeted drug delivery.

[0037] Beneficial Effects

[0038] 1. The pentagalloyl glucose liposomes provided by the present invention can encapsulate pentagalloyl glucose in the vesicles of the liposomes, separate pentagalloyl glucose from the external environment, improve the stability of the encapsulated drug containing pentagalloyl glucose, and enhance the bioavailability of pentagalloyl glucose.

[0039] 2. The pentagalloyl glucose liposomes provided by the present invention can encapsulate pentagalloyl glucose in the vesicles of the liposomes, separate pentagalloyl glucose from the external environment, prolong the action time of the drug to a certain extent, improve the stability of pentagalloyl glucose in the body, and achieve more clear targeted controlled release and other effects.

[0040] 3. The preparation method of the pentagalloyl glucose liposome provided by the invention is simple, the encapsulation rate of pentagalloyl glucose is high, the obtained pentagalloyl glucose liposome has stable properties, and the preparation cycle is short, the cost is low, and it is suitable for large-scale production.

[0041] 4. The encapsulation rate of the pentagalloylglucose liposomes provided by the present invention is above 85%. The high encapsulation rate can effectively delay the oxidation or hydrolysis rate of pentagalloylglucose.

[0042] 5. The pentagalloyl glucose liposomes provided by the present invention have a smaller particle size and can more effectively penetrate into cells and tissues to exert targeted drug effects.

[0043] 6. The pentagalloyl glucose liposomes provided by the present invention have good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is the test result of the particle size of the pentagalloylglucose liposome provided in Example 2.

[0045] Figure 2 The figure is the test result of the stability of the pentagalloylglucose liposomes provided in Example 2. DETAILED DESCRIPTION

[0046] Example 1

[0047] The first aspect of the present embodiment provides a method for preparing pentagalloyl glucose liposomes, the preparation method comprising the following steps:

[0048] S1. adding liposomes into an organic solvent for dissolution to obtain a lipid solution;

[0049] S2. mixing pentagalloylglucose with a phosphate buffer solution to obtain a pentagalloylglucose solution;

[0050] S3. mixing the lipid solution and the pentagalloylglucose solution and stirring to obtain micelles;

[0051] S4. removing the organic solvent in the micelle to obtain a lipid membrane;

[0052] S5. mixing the lipid film with a surfactant and a phosphate buffer solution to obtain a mixed solution;

[0053] S6. Filter the mixed solution to obtain pentagalloylglucose liposomes.

[0054] In the S1, liposomes are added to an organic solvent and magnetically stirred at room temperature of 25° C. and a stirring speed of 600 rpm for 20 minutes to dissolve and obtain a lipid solution.

[0055] The solid-to-liquid ratio of the liposomes to the organic solvent in the S1 is 8:1 mg / mL.

[0056] The added amount of the liposome is 120 mg. The liposome is composed of soybean lecithin and cholesterol, and the mass ratio of the soybean lecithin to cholesterol is 3:1.

[0057] The amount of the organic solvent added in S1 is 15 mL, and the organic solvent is chloroform.

[0058] In the S2, the pentagalloyl glucose and the phosphate buffer solution are stirred and mixed for 30 minutes to obtain a pentagalloyl glucose solution, and the pH of the phosphate buffer solution in the S2 is 7.5.

[0059] The solid-liquid ratio of pentagalloylglucose to phosphate buffer solution in S2 is 5:1 mg / mL, the amount of pentagalloylglucose added is 15 mg, and the amount of phosphate buffer solution added is 3 mL.

[0060] In S3, the lipid solution and the pentagalloylglucose solution are mixed and magnetically stirred at room temperature of 25° C. and a stirring speed of 650 rpm for 2 hours. The amount of the lipid solution added is 15 mL, and the amount of the pentagalloylglucose solution added is 3 mL.

[0061] In the S4, a rotary evaporator is used to remove the organic solvent in the micelle at 40° C. for 1 h to obtain a lipid film.

[0062] In S5, the lipid film obtained in S4 is mixed with 30 mL of phosphate buffer solution, and a mixed solution is obtained by magnetically stirring at room temperature of 25° C. and a stirring speed of 650 rpm for 1.5 h.

[0063] The pH of the phosphate buffer solution in S5 is 7.5.

[0064] In S6, the mixed solution is allowed to stand for 2 hours before filtering, and a microporous filter membrane with a pore size of 0.22 μm is used to filter the particles.

[0065] After the pentagalloyl glucose liposomes are obtained in S6, the pentagalloyl glucose liposomes are stored at 4°C.

[0066] The second aspect of this embodiment provides a pentagalloylglucose liposome, which is prepared by the above-mentioned preparation method of pentagalloylglucose liposome.

[0067] Example 2

[0068] The first aspect of the present embodiment provides a method for preparing pentagalloyl glucose liposomes, the preparation method comprising the following steps:

[0069] S1. adding liposomes into an organic solvent for dissolution to obtain a lipid solution;

[0070] S2. mixing pentagalloylglucose with a phosphate buffer solution to obtain a pentagalloylglucose solution;

[0071] S3. mixing the lipid solution and the pentagalloylglucose solution and stirring to obtain micelles;

[0072] S4. removing the organic solvent in the micelle to obtain a lipid membrane;

[0073] S5. mixing the lipid film with a surfactant and a phosphate buffer solution to obtain a mixed solution;

[0074] S6. Filter the mixed solution to obtain pentagalloylglucose liposomes.

[0075] In S1, liposomes are added into an organic solvent and magnetically stirred at room temperature of 25° C. and a stirring speed of 600 rpm for 20 min to dissolve and obtain a lipid solution.

[0076] The solid-to-liquid ratio of the liposomes to the organic solvent in the S1 is 8:1 mg / mL.

[0077] The added amount of the liposome is 120 mg. The liposome is composed of soybean lecithin and cholesterol, and the mass ratio of the soybean lecithin to cholesterol is 3:1.

[0078] The amount of the organic solvent added in S1 is 15 mL, and the organic solvent consists of chloroform and ether. The amount of chloroform added is 6 mL, and the amount of ether added is 9 mL.

[0079] In the S2, the pentagalloyl glucose and the phosphate buffer solution are stirred and mixed for 30 minutes to obtain a pentagalloyl glucose solution, and the pH of the phosphate buffer solution in the S2 is 7.5.

[0080] The solid-liquid ratio of pentagalloylglucose to phosphate buffer solution in S2 is 5:1 mg / mL, the amount of pentagalloylglucose added is 15 mg, and the amount of phosphate buffer solution added is 3 mL.

[0081] In S3, the lipid solution and the pentagalloylglucose solution are mixed, and magnetic stirring is performed at room temperature of 25° C. and a stirring speed of 650 rpm for 2 hh. The amount of the lipid solution added is 15 mL, and the amount of the pentagalloylglucose solution added is 3 mL.

[0082] In the S4, a rotary evaporator is used to remove the organic solvent in the micelle at 40° C. for 1 h to obtain a lipid film.

[0083] In S5, the lipid film obtained in S4 is mixed with 300 μL of a surfactant and 30 mL of a phosphate buffer solution, and a mixed solution is obtained by magnetically stirring at a room temperature of 25° C. and a stirring speed of 650 rpm for 1.5 h.

[0084] The surfactant in the S5 is Tween-80.

[0085] The pH of the phosphate buffer solution in S5 is 7.5.

[0086] In S6, the mixed solution is allowed to stand for 2 hours before filtering, and a microporous filter membrane with a pore size of 0.22 μm is used to filter the particles.

[0087] After the pentagalloyl glucose liposomes are obtained in S6, the pentagalloyl glucose liposomes are stored at 4°C.

[0088] The second aspect of this embodiment provides a pentagalloylglucose liposome, which is prepared by the above-mentioned preparation method of pentagalloylglucose liposome.

[0089] The third aspect of this embodiment provides an application of pentagalloylglucose liposomes, wherein the pentagalloylglucose liposomes are applied in the field of targeted drug delivery technology.

[0090] The purchase information of the raw materials used in the above-mentioned Examples 1 and 2 is shown in Table a-1.

[0091] Table a-1

[0092] raw material Buy from manufacturers model Soy lecithin Adamas 99%, derived from soybeans, RG cholesterol Adamas 98%, from pig brain, RG Pentagalloylglucose Fujian Ruiboao Technology Co., Ltd. Batch number: 20240425 Twain-80 Adamas Pharmaceutical grade

[0093] Performance Testing

[0094] 1. Determination of encapsulation efficiency:

[0095] (1) After the pentagalloyl glucose liposomes prepared in Example 1 and Example 2 were restored to a temperature of 25° C., 0.5 mL of the pentagalloyl glucose liposomes provided in Example 1 and 1.5 mL of the pentagalloyl glucose liposomes provided in Example 2 were taken, and three sets of parallel tests were performed on Example 2 (each test amount was 0.5 mL). The pentagalloyl glucose lipids were placed in different centrifuge tubes, and then high-purity water for injection was added to make the volume 10 mL, mixed, and centrifuged at 15° C. and 13,000 rpm for 30 min. The supernatant was aspirated, and the supernatant was measured by a spectrophotometer to calculate the concentration of free pentagalloyl glucose in the supernatant; the three sets of parallel tests were performed on Example 2.

[0096] (2) After the pentagalloyl glucose liposomes prepared in Example 1 and Example 2 were restored to a temperature of 25°C, 0.5 mL of the pentagalloyl glucose liposomes provided in Example 1 and 1.5 mL of the pentagalloyl glucose liposomes provided in Example 2 were taken, and three sets of parallel tests were performed on Example 2 (each test amount was 0.5 mL). The pentagalloyl glucose lipids were placed in different centrifuge tubes, and then 4.5 mL of anhydrous ethanol was added and ultrasonicated at room temperature of 25°C and 80W for 10 min to completely demulsify the emulsions. High-purity water for injection was then added to make the volume to 10 mL, mixed, and centrifuged at 15°C at a speed of 13000 rpm for 30 min, and the supernatant was aspirated. The supernatant was measured by a spectrophotometer to calculate the concentration of total pentagalloyl glucose in the supernatant; the three sets of parallel tests were performed on Example 2.

[0097] The encapsulation efficiency of pentagalloylglucose liposome (%) = (the concentration of total pentagalloylglucose - the concentration of free pentagalloylglucose) / the concentration of total pentagalloylglucose * 100%.

[0098] The results of testing the encapsulation efficiency of the pentagalloylglucose liposomes provided in Example 1 are shown in Table 1.

[0099] The results of the three sets of parallel tests on the encapsulation efficiency of the pentagalloylglucose liposomes provided in Example 2 are shown in Table 1.

[0100] 2. Measurement of particle size:

[0101] The particle size of the pentagalloylglucose liposomes provided in Example 2 was measured using a Malvern particle sizer. The parameters set for the Malvern particle sizer were as follows: slot type: DTS0012 disposable cuvette; solvent: distilled water; temperature: 25°C. The particle size measurement results are shown in Figure 1 .

[0102] 3. Determination of the stability of pentagalloyl glucose liposomes:

[0103] (1) The pH of the pentagalloyl glucose liposomes provided in Example 2 was measured using a pH tester, once a day for one week to test the stability of the pentagalloyl glucose liposomes. The specific results are shown in Figure 2 .

[0104] Table 1

[0105]

[0106] It can be seen from the test results in Table 1 that the encapsulation rate of the pentagalloylglucose liposomes provided in Example 2 is above 85%. A high encapsulation rate can effectively slow down the oxidation or hydrolysis rate of pentagalloylglucose.

[0107] pass Figure 1 The test results show that the particle size of the pentagalloylglucose liposomes provided in Example 2 is basically between 10-1000 nm, which is smaller and can more effectively penetrate into cells and tissues to exert targeted drug effects.

[0108] pass Figure 2 The test data in show that during the measurement period of one week, the pH change of the pentagalloyl glucose liposomes provided in Example 2 is small, and it can be judged that the pH stability of the obtained pentagalloyl glucose liposomes is relatively high.

Claims

1. A method for preparing pentagalloyl glucose liposomes, characterized in that: The preparation method comprises the following steps: S1. adding liposomes into an organic solvent for dissolution to obtain a lipid solution; S2. mixing pentagalloylglucose with a phosphate buffer solution to obtain a pentagalloylglucose solution; S3. mixing the lipid solution and the pentagalloylglucose solution and stirring to obtain micelles; S4. removing the organic solvent in the micelle to obtain a lipid membrane; S5. mixing the lipid film with a surfactant and a phosphate buffer solution to obtain a mixed solution; S6. Filter the mixed solution to obtain pentagalloylglucose liposomes.

2. The method for preparing pentagalloyl glucose liposomes according to claim 1, characterized in that: The solid-to-liquid ratio of the liposomes to the organic solvent in the S1 is (7-9):1 mg / mL.

3. The method for preparing pentagalloyl glucose liposomes according to claim 1, characterized in that: The liposome in S1 is selected from one or more combinations of cholesterol, soybean lecithin, and hydrogenated soybean lecithin.

4. The method for preparing pentagalloyl glucose liposomes as claimed in claim 3, characterized in that: The liposome comprises soybean lecithin and cholesterol, and the mass ratio of the soybean lecithin to cholesterol is (2-4):

1.

5. The method for preparing pentagalloyl glucose liposomes according to claim 1, characterized in that: The solid-to-liquid ratio of pentagalloylglucose to phosphate buffer solution in S2 is (1-5): 1 mg / mL.

6. The method for preparing pentagalloyl glucose liposomes according to claim 1, characterized in that: In S3, the lipid solution and the pentagalloylglucose solution are mixed and stirred for 1.5-2 hours.

7. The method for preparing pentagalloyl glucose liposomes according to claim 1, characterized in that: The surfactant in S5 includes one or more combinations of Tween-20, Tween-40, Tween-40, and Tween-80.

8. The method for preparing pentagalloyl glucose liposomes according to claim 1, characterized in that: In the above-mentioned S6, the mixed solution is filtered using a microporous filter membrane with a pore size of 0.1-0.5 μm.

9. A pentagalloyl glucose liposome, characterized in that: The liposome is prepared by the method for preparing pentagalloyl glucose liposomes as described in any one of claims 1 to 8.

10. A use of the pentagalloylglucose liposome as claimed in claim 9, characterized in that: The pentagalloyl glucose liposome is applied to the technical field of targeted drug delivery.

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