Lipid carrier for treating brain glioma as well as preparation method and application of lipid carrier

Abstract

The invention relates to the technical field of pharmaceutical preparations. The invention provides a lipid carrier for treating brain glioma and a preparation method thereof, and the preparation method comprises the following steps: dissolving soybean lecithin, cholesterol, glyceryl monostearate and medicinal components in an organic solvent, then mixing with D-mannose dissolved in absolute ethyl alcohol, and carrying out rotary evaporation to obtain a lipid membrane; adding a phosphate buffer solution into the lipid membrane obtained in the step (1), and stirring to obtain a liposome suspension; and carrying out ultrasonic treatment on the liposome suspension in the step (2), and filtering to obtain the liposome. The D-mannose ligand is utilized to modify the liposome, and the drug effect components adriamycin and isoliquiritigenin are combined for use, so that the liposome stability and the high drug entrapment amount are ensured, meanwhile, the liposome can better act on the glioma part in a targeting manner, and the purpose of synergistic treatment is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and in particular to a lipid carrier for treating brain gliomas, and a preparation method and application thereof. Background Art

[0002] Gliomas are the most common and deadly brain tumors. Due to their invasive growth and high malignancy, clinical treatment effects are very poor. Gene therapy, as a new treatment method, has become a research hotspot in recent years. Among them, liposome drug delivery systems have shown potential in the treatment of gliomas due to their unique physical and chemical properties. However, due to the complex anatomical structure of the central nervous system and the role of the blood-brain barrier, delivery has become a major problem. Although lipid carriers have shown certain potential in the treatment of gliomas, there are still some problems as follows: (1) The existence of the blood-brain barrier makes it difficult for drugs to penetrate, affecting the treatment effect; (2) The current delivery system has low efficiency and it is difficult to achieve efficient drug delivery; (3) How to ensure that the drug has no toxic side effects on normal brain tissue during the process of crossing the blood-brain barrier; etc.

[0003] In summary, it is still very important to develop efficient and safe central nervous system delivery carriers to efficiently deliver loaded drugs to the lesion site, which is of great significance for the future treatment of brain gliomas. Summary of the invention

[0004] The purpose of the present invention is to provide a lipid carrier for treating brain glioma. By using the active ingredients doxorubicin and isoliquiritigenin and modifying the liposomes with D-mannose ligands, the stability of the liposomes and the high drug loading capacity can be ensured while better targeting the brain glioma site can be achieved, thereby achieving the purpose of synergistic treatment.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing a lipid carrier for treating brain glioma, comprising the following steps:

[0007] (1) dissolving soybean lecithin, cholesterol, monostearate glyceryl and a medicinal ingredient in an organic solvent, then mixing with D-mannose dissolved in anhydrous ethanol, and rotary evaporating to obtain a lipid film;

[0008] (2) adding phosphate buffer to the lipid film of step (1), and stirring to obtain a liposome suspension;

[0009] (3) After ultrasonication and filtration of the liposome suspension described in step (2), the lipid carrier for treating brain glioma is obtained.

[0010] Preferably, the medicinal ingredients in step (1) include doxorubicin and / or isoliquiritigenin.

[0011] Preferably, when the active ingredients are doxorubicin and isoliquiritigenin, the mass ratio of doxorubicin to isoliquiritigenin is (1-3):(0.5-1.5).

[0012] Preferably, the mass ratio of soybean lecithin, cholesterol, glyceryl monostearate and medicinal ingredient in step (1) is (18-25):(4-8):(6-10):(2-4).

[0013] Preferably, the mass ratio of soybean lecithin to D-mannose is (9-15):1.

[0014] Preferably, the temperature of the rotary evaporation in step (1) is 42-52° C., and the time of the rotary evaporation is 10-15 min.

[0015] Preferably, the power of the ultrasound in step (3) is 160-220 W, and the time of the ultrasound is 60-140 s.

[0016] Preferably, the filtration specification in step (3) is 0.20-0.25 μm.

[0017] The present invention also provides a lipid carrier prepared by the preparation method and used for treating brain glioma.

[0018] The present invention also provides application of the lipid carrier in preparing a product for treating brain glioma.

[0019] By adopting the above technical solution, the present invention has the following beneficial effects:

[0020] In the technical solution of the present invention, D-mannose is used as a ligand to modify the surface of the drug-loaded liposome, which not only enhances the ability of the drug-loaded liposome to target tumor cells, but also effectively improves the transport capacity across the blood-brain barrier; the flavonoid compound isoliquiritigenin in licorice and doxorubicin are used as active ingredients, and the combination of the two achieves a synergistic or additive effect in treating brain gliomas, and also reduces the amount of doxorubicin used and its toxic side effects. Therefore, the present invention can better target the brain glioma site while ensuring the stability of the liposome and the high drug loading capacity through the combined action of ligand modification and composite active ingredients, effectively release the drug, and achieve the purpose of efficient and safe treatment. DETAILED DESCRIPTION

[0021] The present invention provides a method for preparing a lipid carrier for treating brain glioma, comprising the following steps:

[0022] (1) dissolving soybean lecithin, cholesterol, monostearate glyceryl and a medicinal ingredient in an organic solvent, then mixing with D-mannose dissolved in anhydrous ethanol, and rotary evaporating to obtain a lipid film;

[0023] (2) adding phosphate buffer to the lipid film of step (1), and stirring to obtain a liposome suspension;

[0024] (3) After ultrasonication and filtration of the liposome suspension described in step (2), the lipid carrier for treating brain glioma is obtained.

[0025] In the present invention, soybean lecithin, cholesterol, monostearate glyceryl and medicinal ingredients are dissolved in an organic solvent and mixed evenly. The organic solvent of the present invention is preferably anhydrous ethanol. The mass ratio of soybean lecithin, cholesterol, monostearate glyceryl and medicinal ingredients of the present invention is preferably (18-25): (4-8): (6-10): (2-4), further preferably (19-23): (5-7): (7-9): (2.5-3.5), and further preferably 20: 6: 8: 3. The medicinal ingredients of the present invention are preferably doxorubicin and / or isoliquiritigenin, and further preferably doxorubicin and isoliquiritigenin. When the medicinal ingredients of the present invention are doxorubicin and isoliquiritigenin, the mass ratio of doxorubicin and isoliquiritigenin is preferably (1-3): (0.5-1.5), further preferably (1.5-2.7): (0.8-1.2), and further preferably 2: 1.

[0026] In the present invention, D-mannose is dissolved in anhydrous ethanol, and then mixed with the above mixture, and after uniform mixing, the lipid film is obtained by rotary evaporation. The mass ratio of soybean lecithin to D-mannose in the present invention is preferably (9-15):1, further preferably (10-14):1, and further preferably 12.5:1. The rotary evaporation of the present invention is preferably carried out in a water bath environment, and the temperature of the water bath is preferably 42-52°C, further preferably 43-50°C, and further preferably 47°C; the time of the rotary evaporation is preferably 10-15min, further preferably 11-14min, and further preferably 13min.

[0027] In the present invention, phosphate buffer is added to the prepared lipid film, and then stirred to obtain a liposome suspension. The present invention preferably blows an appropriate amount of nitrogen into the lipid film before adding the phosphate buffer, which can better protect the lipid film. The volume ratio of the total mass of soybean lecithin, cholesterol, monostearate glyceryl and medicinal ingredients to phosphate buffer in the present invention is preferably 1mg: (7-10) mL, further preferably 1mg: (7.5-9) mL, and further preferably 1mg: 8mL. The stirring of the present invention is preferably carried out in a water bath environment, and the temperature of the water bath is preferably 43-50°C, further preferably 45-49°C, and further preferably 46°C. The stirring of the present invention is preferably magnetic stirring, and the stirring time is preferably 18-28min, further preferably 20-27min, and further preferably 25min.

[0028] In the present invention, the liposome suspension is ultrasonicated and filtered to obtain the lipid carrier for treating brain glioma. The power of the ultrasound in the present invention is preferably 160-220W, further preferably 170-200W, and further preferably 180W; the time of the ultrasound is preferably 60-140s, further preferably 80-130s, and further preferably 110s. The filtration in the present invention is preferably carried out using a microporous filter membrane, and the specification of the microporous filter membrane is preferably 0.20-0.25μm, further preferably 0.21-0.24μm, and further preferably 0.22μm.

[0029] The present invention also provides a lipid carrier prepared by the preparation method and used for treating brain glioma.

[0030] The present invention also provides application of the lipid carrier in preparing a product for treating brain glioma.

[0031] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0032] Example 1

[0033] A method for preparing a lipid carrier for treating brain glioma, comprising the following steps:

[0034] (1) dissolving soybean lecithin, cholesterol, monostearate glyceryl and an active ingredient in anhydrous ethanol at a mass ratio of 20:6:8:3, wherein the active ingredient is doxorubicin and isoliquiritigenin at a mass ratio of 2:1, and then mixing with D-mannose dissolved in anhydrous ethanol, wherein the mass ratio of soybean lecithin to D-mannose is 12.5:1; after mixing evenly, performing rotary evaporation in a water bath at 47° C. for 13 minutes to obtain a lipid film;

[0035] (2) blowing an appropriate amount of nitrogen into the lipid film, and then adding phosphate buffer in a volume ratio of 1 mg:8 mL of the total mass of soybean lecithin, cholesterol, monostearate glyceryl, and the active ingredient to phosphate buffer, and stirring the mixture under magnetic stirring in a water bath at 46° C. for 25 min to obtain a liposome suspension;

[0036] (3) The liposome suspension was ultrasonically treated at 180 W for 110 s, and then filtered through a 0.22 μm microporous filter membrane to obtain a lipid carrier for treating brain glioma.

[0037] Example 2

[0038] A method for preparing a lipid carrier for treating brain glioma, comprising the following steps:

[0039] (1) Soybean lecithin, cholesterol, monostearate glyceryl and an active ingredient are dissolved in anhydrous ethanol in a mass ratio of 18:8:6:2, wherein the active ingredient is doxorubicin and isoliquiritigenin in a mass ratio of 1.5:0.5, and then mixed with D-mannose dissolved in anhydrous ethanol, wherein the mass ratio of soybean lecithin to D-mannose is 10:1; after mixing evenly, the mixture is subjected to rotary evaporation in a water bath at 50° C. for 10 min to obtain a lipid film;

[0040] (2) blowing an appropriate amount of nitrogen into the lipid film, and then adding phosphate buffer according to the volume ratio of the total mass of soybean lecithin, cholesterol, monostearate glyceryl, and the active ingredient to phosphate buffer of 1 mg:7 mL, and magnetically stirring for 27 minutes in a water bath at 45° C. to obtain a liposome suspension;

[0041] (3) The liposome suspension was ultrasonically treated at 200 W for 80 s, and then filtered through a 0.22 μm microporous filter membrane to obtain a lipid carrier for treating brain glioma.

[0042] Example 3

[0043] A method for preparing a lipid carrier for treating brain glioma, comprising the following steps:

[0044] (1) Soybean lecithin, cholesterol, monostearate glyceryl and an active ingredient are dissolved in anhydrous ethanol in a mass ratio of 25:4:10:2.5, wherein the active ingredient is doxorubicin and isoliquiritigenin in a mass ratio of 1.5:1, and then mixed with D-mannose dissolved in anhydrous ethanol, wherein the mass ratio of soybean lecithin to D-mannose is 14:1; after mixing evenly, the mixture is subjected to rotary evaporation in a water bath at 43° C. for 15 minutes to obtain a lipid film;

[0045] (2) blowing an appropriate amount of nitrogen into the lipid film, and then adding phosphate buffer according to the volume ratio of the total mass of soybean lecithin, cholesterol, monostearate glyceryl, and active ingredients to phosphate buffer of 1 mg:10 mL, and stirring magnetically in a water bath at 49° C. for 20 min to obtain a liposome suspension;

[0046] (3) The liposome suspension was ultrasonically treated at 160 W for 140 s, and then filtered through a 0.22 μm microporous filter membrane to obtain a lipid carrier for treating brain glioma.

[0047] Example 4

[0048] A method for preparing a lipid carrier for treating brain glioma, comprising the following steps:

[0049] (1) dissolving soybean lecithin, cholesterol, monostearate glyceryl and doxorubicin in anhydrous ethanol at a mass ratio of 20:6:8:3, and then mixing with D-mannose dissolved in anhydrous ethanol, wherein the mass ratio of soybean lecithin to D-mannose is 12.5:1; after mixing evenly, performing rotary evaporation in a water bath at 47° C. for 13 min to obtain a lipid film;

[0050] (2) blowing an appropriate amount of nitrogen into the lipid film, and then adding phosphate buffer in a volume ratio of 1 mg:8 mL of the total mass of soybean lecithin, cholesterol, monostearate glyceryl, and the active ingredient to phosphate buffer, and stirring the mixture under magnetic stirring in a water bath at 46° C. for 25 min to obtain a liposome suspension;

[0051] (3) The liposome suspension was ultrasonically treated at 180 W for 110 s, and then filtered through a 0.22 μm microporous filter membrane to obtain a lipid carrier for treating brain glioma.

[0052] Example 5

[0053] A method for preparing a lipid carrier for treating brain glioma, comprising the following steps:

[0054] (1) dissolving soybean lecithin, cholesterol, monostearate glyceryl and isoliquiritigenin in anhydrous ethanol at a mass ratio of 20:6:8:3, and then mixing with D-mannose dissolved in anhydrous ethanol, wherein the mass ratio of soybean lecithin to D-mannose is 12.5:1; after mixing evenly, performing rotary evaporation in a water bath at 47° C. for 13 min to obtain a lipid film;

[0055] (2) blowing an appropriate amount of nitrogen into the lipid film, and then adding phosphate buffer in a volume ratio of 1 mg:8 mL of the total mass of soybean lecithin, cholesterol, monostearate glyceryl, and the active ingredient to phosphate buffer, and stirring the mixture under magnetic stirring in a water bath at 46° C. for 25 min to obtain a liposome suspension;

[0056] (3) The liposome suspension was ultrasonically treated at 180 W for 110 s, and then squeezed through a 0.22 μm microporous filter membrane for filtration to obtain a lipid carrier for treating brain glioma.

[0057] Test Example 1

[0058] 20 mg soybean lecithin, 6 mg cholesterol, 8 mg monostearate glyceryl, 2 mg doxorubicin and 1 mg isoliquiritigenin were dissolved in anhydrous ethanol and then mixed with 1.6 mg D-mannose dissolved in anhydrous ethanol; after uniform mixing, the mixture was evaporated in a water bath at 47 ° C for 13 min to obtain a lipid film. An appropriate amount of nitrogen was blown into the lipid film, and then 296 mL of phosphate buffer was added. After magnetic stirring for 25 min in a water bath at 46 ° C, a liposome suspension was obtained. The liposome suspension was ultrasonically treated at 180 W for 110 s, and then filtered through a 0.22 μm microporous filter membrane by extrusion to obtain a lipid carrier for the treatment of brain glioma.

[0059] (I) Encapsulation efficiency

[0060] The lipid carrier prepared as above was used as the test group, and a control group lipid carrier was prepared based on this process.

[0061] Control group 1: lipid carrier without D-mannose ligand modification, other operations were the same as those of the experimental group.

[0062] Control group 2: Only 8 mg of soybean lecithin was used, and other operations were the same as the experimental group.

[0063] The lipid carriers of the test group and the lipid carriers of the control group were placed in 10 mL volumetric flasks, and 10 volumes of ethanol were used to detect the concentrations of doxorubicin and isoliquiritigenin, which were recorded as A1 and A2, respectively. The lipid carriers of the test group and the lipid carriers of the control group were passed through a shephedex G-50 column, eluted with a pH 7.4 PBS buffer to remove free isoliquiritigenin, then placed in a dialysis bag and dialyzed in a pH 7.4 PBS buffer to remove free doxorubicin, then placed in a 10 mL volumetric flask, destroyed with 10 volumes of ethanol, and the concentrations of doxorubicin and isoliquiritigenin were detected, which were recorded as B1 and B2, respectively.

[0064] Encapsulation efficiency (%) = B1 / A1×100% or B2 / A2×100%

[0065] Table 1 Encapsulation efficiency of different lipid carriers

[0066]

[0067]

[0068] It can be seen from Table 1 that the lipid carrier prepared by the technical solution of the present invention has a relatively good encapsulation efficiency for doxorubicin and isoliquiritigenin.

[0069] (II) In vitro cell uptake experiment

[0070] U87 human glioma cells in the logarithmic growth phase (purchased from Wuhan Pronocell Life Science Co., Ltd.) were plated at 1×10 5 The cells were inoculated in a 24-well plate containing 10% serum. After culturing for 24 hours, the sample test group liposome suspension, the control group 1 liposome suspension and the control group 2 liposome suspension were added to the well plate, 100 μL was added to each well, and the cells were incubated in a CO2 incubator for 2 hours. The culture medium was discarded, and an appropriate amount of PBS was added to wash 3 times. 1 mL of 1% triton-100 was added to lyse the cells, and the cells were lysed at 4°C in the dark for more than 0.5 hour. After complete lysis, 200 μL of each sample from each group was taken and the fluorescence intensity was measured by a fluorescence spectrophotometer at Ex=465nm and Em=502nm to calculate the uptake efficiency. The results are shown in Table 2.

[0071] Table 2 In vitro cellular uptake efficiency of different lipid carriers

[0072] Group Cell uptake efficiency (%) Experimental Group 128.9 Control group 1 56.3 Control group 2 120.1

[0073] It can be seen from Table 2 that the lipid carrier modified with D-mannose ligand in the present invention has a significant uptake effect on U87 cells and can promote cell uptake.

[0074] (III) Inhibitory effect on U87 human glioma cells

[0075] U87 human glioma cells in the logarithmic growth phase were cultured at 1×10 5 Cells / well were inoculated in a 96-well plate and cultured at 37°C and 5% CO2 for 24 hours. Then, 10 μL of a series of drug-loaded lipid carrier solutions prepared with pH 7.4 PBS buffer were added to each well of the 96-well plate. The concentrations of doxorubicin in each treatment group were 0.1, 0.5, 1, 2.5, 5 or 10 μM. At the same time, an equal volume of PBS buffer was used to replace the drug-loaded lipid carrier solution to set up a blank control group. The plates were cultured at 37°C and 5% CO2 for 48 hours, and then the culture supernatant was discarded. The absorbance OD value of each well at 540 nm was determined by the SRB method.

[0076] Cell survival rate = OD value of experimental group / OD value of blank control group × 100%.

[0077] Table 3 Effects of different groups of lipid carriers on cell activity

[0078]

[0079]

[0080] It can be seen from Table 3 that the drug-loaded lipid carrier prepared by the present invention has a significant inhibitory effect on U87 human brain glioma cells.

[0081] In summary, it can be seen that the present invention uses the active ingredients doxorubicin and isoliquiritigenin and modifies the liposomes with D-mannose ligands, thereby achieving better targeting to the brain glioma site while ensuring the stability of the liposomes and a high drug loading capacity, thereby achieving the purpose of synergistic treatment.

[0082] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a lipid carrier for treating brain glioma, characterized in that: The steps include: (1) dissolving soybean lecithin, cholesterol, monostearate glyceryl and a medicinal ingredient in an organic solvent, then mixing with D-mannose dissolved in anhydrous ethanol, and rotary evaporating to obtain a lipid film; (2) adding phosphate buffer to the lipid film of step (1), and stirring to obtain a liposome suspension; (3) After ultrasonication and filtration of the liposome suspension described in step (2), the lipid carrier for treating brain glioma is obtained.

2. The preparation method according to claim 1, characterized in that: The medicinal ingredients in step (1) include doxorubicin and / or isoliquiritigenin.

3. The preparation method according to claim 2, characterized in that: When the active ingredients are doxorubicin and isoliquiritigenin, the mass ratio of doxorubicin to isoliquiritigenin is (1-3):(0.5-1.5).

4. The preparation method according to claim 1, characterized in that: The mass ratio of soybean lecithin, cholesterol, glyceryl monostearate and medicinal ingredient in step (1) is (18-25):(4-8):(6-10):(2-4); The mass ratio of soybean lecithin to D-mannose is (9-15):

1.

5. The preparation method according to claim 1, characterized in that: The temperature of the rotary evaporation in step (1) is 42-52° C., and the time of the rotary evaporation is 10-15 min.

6. The preparation method according to claim 1, characterized in that: The power of the ultrasound in step (3) is 160-220W, and the time of the ultrasound is 60-140s.

7. The preparation method according to claim 1, characterized in that: The filtration specification in step (3) is 0.20-0.25 μm.

8. A lipid carrier for treating brain glioma prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the lipid carrier according to claim 8 in preparing a product for treating brain glioma.

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