A liposome and its application, a liposome preparation and its preparation method and application

Abstract

The present invention belongs to the technical field of liposome pharmaceutical preparations, and specifically relates to a liposome and its application, a liposome preparation and its preparation method and application. The present invention provides a liposome, wherein the liposome membrane is modified with a glutathione group and an indole residue. The liposome provided by the present invention has excellent transmembrane transport ability, efficiently penetrates the blood-brain barrier and can be taken up by glioma cells, while exhibiting excellent endosomal escape properties, thereby effectively solving the technical problem of low blood-brain barrier permeability during the treatment of gliomas with temozolomide. At the same time, the present invention provides a liposome preparation that improves delivery efficiency and anti-tumor efficacy, has an inhibitory effect on both human and mouse glioma cells, shows a wide range of clinical application potential, and provides a new treatment plan for glioma patients with high efficiency and low toxicity.

Description

Technical Field

[0001] The present invention belongs to the technical field of liposome pharmaceutical preparations, and in particular relates to a liposome and an application thereof, a liposome preparation and a preparation method and an application thereof. Background Art

[0002] Glioblastoma (GBM) is one of the most common primary malignant tumors of the central nervous system and is highly invasive and heterogeneous. Its rapid proliferation, diffuse growth, and unclear boundaries with normal brain tissue make it difficult to treat. Traditional treatments, such as surgery, radiotherapy, and chemotherapy, are often limited in effectiveness due to the blood-brain barrier (BBB). The existence of the BBB makes it difficult for many chemotherapy drugs to effectively reach the tumor area, thereby limiting the effectiveness of treatment.

[0003] Temozolomide (TMZ), a standard chemotherapy drug for glioma, exerts its anti-tumor effects primarily by alkylating DNA. Although TMZ can prolong survival in some patients, its efficacy is limited by the low permeability of the blood-brain barrier, which reduces its clinical effectiveness. Summary of the Invention

[0004] The present invention aims to provide a liposome and its application, a liposome preparation and its preparation method and application. The liposome provided by the present invention has excellent transmembrane transport ability, can efficiently penetrate the blood-brain barrier and be taken up by glioma cells, and at the same time exhibits excellent endosomal escape properties, thereby effectively solving the technical problem of low blood-brain barrier permeability during temozolomide treatment of gliomas.

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

[0006] The invention provides a liposome, wherein the liposome membrane is modified with a glutathione group and an indole residue.

[0007] Preferably, the liposomes include the following raw materials in parts by weight:

[0008] 2-8 parts of indole-oleamine, 20-40 parts of DSPE-PEG-glutathione, 80-120 parts of phospholipids, and 2-6 parts of cholesterol;

[0009] The indole-oleylamine is prepared by condensing oleylamine and indole-2-carboxylic acid.

[0010] Preferably, the phospholipids include one or more of soybean lecithin, egg yolk lecithin, hydrogenated lecithin and synthetic lecithin;

[0011] The raw materials for preparing the DSPE-PEG-glutathione include polyethylene glycol, and the weight-average molecular weight of the polyethylene glycol is 1000-10000 Da.

[0012] The present invention provides the use of the liposome described in the above technical solution in preparing a brain-targeted liposome preparation.

[0013] The present invention provides a liposome preparation, comprising the liposome described in the above technical solution;

[0014] The invention also includes drug molecules encapsulated in the vesicles of the liposome, wherein the drug molecules include glutamic acid-modified temozolomide and elemene.

[0015] Preferably, the glutamic acid-modified temozolomide comprises one or more of the following chemical structures:

[0016] .

[0017] Preferably, the elemene includes one or more of α-elemene, β-elemene, γ-elemene and δ-elemene.

[0018] Preferably, the liposome preparation comprises the following raw materials in parts by weight:

[0019] 2-8 parts of indole-oleamine, 20-40 parts of DSPE-PEG-glutathione, 20-200 parts of glutamic acid-modified temozolomide, 10-40 parts of elemene, 80-120 parts of phospholipids and 2-6 parts of cholesterol.

[0020] The present invention provides a method for preparing the liposome preparation described in the above technical solution, comprising the following steps:

[0021] dissolving the raw materials for preparing the liposome preparation in an organic solvent to obtain a drug mixed solution;

[0022] The organic solvent is removed from the drug mixed solution, and then hydrated with a hydration medium to obtain the liposome preparation.

[0023] The present invention provides the use of the liposome preparation described in the above technical solution or the liposome preparation prepared by the preparation method described in the above technical solution in the preparation of brain-targeted anticancer drugs.

[0024] The present invention provides a liposome whose membrane is modified with a glutathione group and an indole residue. The present invention utilizes glutathione and indole residues as dual targeting groups on the liposome membrane. The glutathione group enables the liposome to better enter tumor cells via glutathione transporters; the indole residue facilitates more efficient transport of small molecule drugs (temozolomide and elemene) through endothelial cells, thereby penetrating the blood-brain barrier. Furthermore, the indole residue disrupts the membrane structure of tumor cells and, upon entry into lysosomes, inserts into the lysosomal lipid bilayer, disrupting the lysosomes and enhancing lysosomal escape. The synergistic effect of the indole and glutathione groups further enhances endocytosis of the liposomes by endothelial cells and their ability to translocate into the endothelium. In summary, the liposomes provided by the present invention possess excellent transmembrane transport capabilities, can efficiently penetrate the blood-brain barrier and be taken up by glioma cells, and exhibit excellent endosomal escape properties, effectively addressing the technical issue of low blood-brain barrier permeability during temozolomide treatment of gliomas.

[0025] The present invention provides a liposome preparation, comprising the liposomes described in the above technical solution; and further comprising drug molecules encapsulated in the vesicles of the liposomes, wherein the drug molecules include glutamic acid-modified temozolomide and elemene. The present invention, through the synergistic effect of indole and glutathione modification, the liposome preparation has the characteristics of efficient glioma targeting, efficient penetration of the blood-brain barrier, transmembrane transport and endosomal escape. On the other hand, the present invention, through the compounding of glutamic acid-modified temozolomide prodrug and elemene, has a highly efficient synergistic effect, which reduces the IC value of the combined treatment of the two. 50 It also effectively reduces the toxic side effects of temozolomide and the irritation of elemene, while enhancing the therapeutic effect and safety, significantly promoting targeting efficiency, significantly reducing tumor cell proliferation and promoting cell apoptosis, while improving delivery efficiency and anti-tumor efficacy, and has an inhibitory effect on both human and mouse glioma cells, showing a wide range of clinical application potential, and providing a new, efficient and low-toxic treatment option for glioma patients.

[0026] The present invention provides a method for preparing the liposome preparation described in the above technical solution. The present invention adopts a thin film hydration method to prepare the liposome preparation. The obtained liposome preparation has uniform particle size, good dispersibility, good stability, simple preparation process, strong repeatability, and is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a particle size distribution diagram of the brain-targeted liposome preparation loaded with elemene and temozolomide prodrug molecules prepared in the present invention;

[0028] Figure 2 To evaluate the blood compatibility of the brain-targeted liposome preparation loaded with elemene and temozolomide prodrug molecules prepared by the present invention;

[0029] Figure 3 To evaluate the anti-nonspecific protein adsorption of the brain-targeted liposome preparation loaded with elemene and temozolomide prodrug molecules prepared by the present invention;

[0030] Figure 4 The cytotoxicity of the brain-targeted liposome preparation loaded with elemene and temozolomide prodrug molecules prepared by the present invention on U-87 MG (human brain astroglioma cells) and GL261 (mouse glioma cells) was evaluated;

[0031] Figure 5 The cellular uptake efficiency of the brain-targeted liposome preparation loaded with elemene and temozolomide prodrug molecules prepared by the present invention;

[0032] Figure 6 The lysosomal escape efficiency of the brain-targeted liposome preparation loaded with elemene and temozolomide prodrug molecules prepared by the present invention in U-87 MG (human brain astroglioma cells) and GL261 (mouse glioma cells);

[0033] Figure 7 The lysosomal escape efficiency of the brain-targeted liposome preparation loaded with elemene and temozolomide prodrug molecules prepared by the present invention in bEnd.3 (mouse brain endothelial cells);

[0034] Figure 8 To evaluate the in vitro blood-brain barrier permeability of the brain-targeted liposome preparation loaded with elemene and temozolomide prodrug molecules prepared by the present invention;

[0035] Figure 9 In vivo distribution imaging and in vitro imaging of the brain-targeted liposome preparation loaded with elemene and temozolomide prodrug molecules prepared by the present invention;

[0036] Figure 10 The invention discloses the effect of the brain-targeted liposome preparation loaded with elemene and temozolomide prodrug molecules on treating glioma in vivo.

[0037] Figure 11 This is the synthetic route of DSPE-PEG-glutathione;

[0038] Figure 12 This is a synthetic route for indole-oleylamine. DETAILED DESCRIPTION

[0039] The invention provides a liposome, wherein the liposome membrane is modified with a glutathione group and an indole residue.

[0040] In the present invention, the glutathione group is derived from the glutathione group of DSPE-PEG-glutathione. In the present invention, the indole residue is derived from the indole group of indole-oleamine. The indole-oleamine is prepared by the condensation reaction of oleylamine and indole-2-carboxylic acid.

[0041] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0042] In the present invention, the indole residue can destroy the integrity of the cell membrane, allowing the indole-functionalized liposome preparation to better enter the cell, thereby achieving the delivery of small molecule drugs (elemene and temozolomide).

[0043] In the present invention, the liposome preferably comprises the following raw materials in parts by weight:

[0044] 2-8 parts of indole-oleamine, 20-40 parts of DSPE-PEG-glutathione, 80-120 parts of phospholipids, and 2-6 parts of cholesterol;

[0045] The indole-oleylamine is prepared by condensing oleylamine and indole-2-carboxylic acid.

[0046] The raw materials for preparing the liposomes provided by the present invention include 2 to 8 parts by mass, preferably 3 to 7 parts, more preferably 4 to 6 parts, and in an embodiment, 5.4 parts of indole-oleamine. The indole-oleamine is prepared by condensing oleylamine and indole-2-carboxylic acid.

[0047] In the present invention, the preparation method of indole-oleylamine preferably comprises the following steps:

[0048] Indole-2-carboxylic acid, 1-hydroxybenzotriazole (HOBT), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and a first organic solvent are mixed for activation to obtain an activation solution;

[0049] The activation solution, oleylamine and a second organic solvent are mixed to carry out a condensation reaction to obtain the indole-oleylamine.

[0050] In the present invention, indole-2-carboxylic acid, HOBT, EDC, and a first organic solvent are mixed for activation to obtain an activated solution. In the present invention, the molar ratio of indole-2-carboxylic acid to oleylamine is preferably 1.5:1. The molar ratio of HOBT to oleylamine is preferably 1:1, and the molar ratio of EDC to oleylamine is preferably 1:1. The first organic solvent is preferably anhydrous dichloromethane. The activation is performed in a protective gas atmosphere, preferably nitrogen. The activation is preferably performed in an ice bath, and the activation time is preferably 30 minutes.

[0051] In an embodiment of the present invention, the chemical structure of the indole-oleylamine is as follows:

[0052] .

[0053] After obtaining the activation solution, the present invention combines the activation solution, oleylamine, and a second organic solvent for a condensation reaction to produce the indole-oleylamine. In the present invention, the second organic solvent is preferably anhydrous dichloromethane. The present invention preferably dissolves the oleylamine in the second organic solvent to produce an oleylamine solution, and then mixes the activation solution and the oleylamine solution. The condensation reaction is preferably carried out at room temperature. The condensation reaction time is preferably 48 hours. The formation of the product during the condensation reaction is preferably detected by TLC. After the condensation reaction is completed, the present invention preferably removes the organic solvent from the resulting condensation reaction solution to produce a mixture; the mixture is redissolved in chloroform and extracted sequentially with an aqueous bicarbonate solution and a saturated sodium chloride solution to produce an extracted organic phase; the extracted organic phase is concentrated and separated by column chromatography to produce the indole-oleylamine. The organic solvent removal method is preferably rotary evaporation. The sodium bicarbonate aqueous solution preferably has a mass content of 5%. The concentration is preferably rotary evaporation. The mobile phase used in the column chromatography separation is a mixed solvent of n-hexane and ethyl acetate, and the volume ratio of n-hexane to ethyl acetate in the mixed solvent is preferably 4:1.

[0054] Based on the weight of the indole-oleylamine, the raw materials for preparing the liposomes provided herein include 20-40 parts, preferably 25-35 parts, and in embodiments, 31.4 parts, of DSPE-PEG-glutathione. In the present invention, the raw materials for preparing the DSPE-PEG-glutathione preferably include phosphoethanolamine phospholipids (DSPE), polyethylene glycol (PEG), and glutathione. The weight-average molecular weight of the PEG used in preparing the DSPE-PEG-glutathione is preferably 1,000 to 10,000 Da.

[0055] In the present invention, the DSPE-PEG-glutathione is preferably synthesized by a Michael-type addition reaction. In a specific embodiment of the present invention, the DSPE-PEG-glutathione is prepared according to the specific method disclosed in "Optimization of glutathione conjugation to liposomes quantified with a validated HPLC assay" (Joy N. Reginald-Opara, Darren Svirskis, Simon J. O'Carroll, Sreevalsan Sreebhavan, Justin M. Dean, Zimei Wu, International Journal of Pharmaceutics 567 (2019) 118451).

[0056] As a specific embodiment of the present invention, the chemical structural formula of the DSPE-PEG-glutathione is as follows:

[0057] .

[0058] Based on the mass fraction of the indole-oleylamine, the raw materials for preparing the liposomes provided by the present invention include 80-120 parts of phospholipids, preferably 90-100 parts, more preferably 95-105 parts, and in the embodiment, 100 parts. In the present invention, the phospholipids preferably include one or more of soybean lecithin, egg yolk lecithin, hydrogenated lecithin, and synthetic lecithin. The hydrogenated lecithin preferably includes hydrogenated soybean lecithin and / or hydrogenated egg yolk lecithin. The synthetic lecithin preferably includes synthetic lecithin. The synthetic lecithin is a phospholipid synthesized by a chemical method.

[0059] Based on the mass fraction of the indole-oleylamine, the raw material for preparing the liposome provided by the present invention includes 2 to 6 parts of cholesterol, preferably 3 to 5 parts, and in the embodiment, it can be 4 parts.

[0060] The present invention provides a method for preparing the liposomes described in the above technical solution, preferably comprising the following steps:

[0061] dissolving the raw materials for preparing the liposome in a third organic solvent to obtain a mixed solution;

[0062] The organic solvent is removed from the mixed solution, and then the mixed solution is hydrated with a hydration medium to obtain the liposome.

[0063] In the present invention, the raw materials for preparing the liposomes are dissolved in a third organic solvent to obtain a mixed solution. In the present invention, the third organic solvent preferably comprises one or more of chloroform, ethanol, dichloromethane, and methanol. The ratio of indole-oleylamine to the third organic solvent is preferably (2-8) mg:100 mL.

[0064] After obtaining a mixed solution, the present invention removes the organic solvent from the mixed solution and then hydrates it with a hydration medium to obtain the liposomes. In the present invention, the method for removing the organic solvent is preferably rotary evaporation. After removing the organic solvent from the mixed solution, a lipid film is obtained. In the present invention, the lipid film and the hydration medium are preferably mixed for hydration. The hydration medium preferably comprises one or more of water, physiological saline, and a phosphate buffer solution with a pH of 7.4. The water is preferably deionized water. The hydration is preferably performed under stirring, at a temperature of 50°C, and for a time of 0.5 to 1 hour. After completion of the hydration, a hydrated solution is obtained. The present invention also preferably includes ultrasonically dispersing the hydrated solution and then filtering it to obtain the liposomes. The ultrasonic dispersion is preferably performed using a probe sonicator. The ultrasonic dispersion conditions preferably include: an ultrasonication time of preferably 20 to 40 minutes, an ultrasonication cycle of 5 seconds, an ultrasonication rest period of 5 seconds, an ultrasonication temperature of ≤70°C, and an ultrasonication power of preferably 225 to 300 W. A 0.45 μm water filter membrane or a 0.22 μm water filter membrane is preferably used for filtration.

[0065] The present invention provides the use of the liposomes described in the above technical solution in the preparation of a brain-targeted liposome preparation. In the present invention, the brain-targeted liposome preparation preferably includes a targeted drug for treating brain glioma.

[0066] The present invention provides a liposome preparation, comprising the liposome described in the above technical solution;

[0067] The invention also includes drug molecules encapsulated in the vesicles of the liposome, wherein the drug molecules include glutamic acid-modified temozolomide and elemene.

[0068] The liposome preparation provided by the present invention includes the liposome described in the above technical solution.

[0069] The liposome preparation provided by the present invention further comprises drug molecules encapsulated in the liposome vesicles, wherein the drug molecules comprise glutamic acid-modified temozolomide (Glu-TMZ) and elemene.

[0070] In the present invention, the glutamic acid-modified temozolomide preferably includes one or more of glutamic acid-temozolomide, second-generation dendrimer glutamic acid-temozolomide and third-generation dendrimer glutamic acid-temozolomide.

[0071] In the present invention, the compound structures of the glutamic acid-temozolomide, the second-generation dendrimer glutamic acid-temozolomide, and the third-generation dendrimer glutamic acid-temozolomide are as follows:

[0072] .

[0073] In the present invention, the glutamic acid-modified temozolomide is preferably prepared by a condensation reaction between a glutamic acid starting material and carboxylated temozolomide (TMZ-COOH). The glutamic acid starting material preferably comprises glutamic acid, a second-generation dendrimer glutamic acid, or a third-generation dendrimer glutamic acid, wherein the amine group is exposed and the carboxyl group is protected. The molar ratio of the carboxyl group of the carboxylated temozolomide to the amine group of the glutamic acid starting material is preferably 2:1.

[0074] In the present invention, the preparation method of the glutamic acid-modified temozolomide preferably comprises the following steps:

[0075] Mixing TMZ-COOH, HOBT, EDC and a fourth organic solvent for activation to obtain an activation solution;

[0076] The activation solution, the glutamic acid preparation raw material and the fifth organic solvent are mixed to carry out a condensation reaction to obtain the glutamic acid-modified temozolomide.

[0077] In the present invention, TMZ-COOH, HOBT, EDC, and a fourth organic solvent are mixed for activation to obtain an activated solution. In the present invention, the molar ratio of the carboxyl groups of the carboxylated temozolomide to the amine groups of the glutamic acid preparation raw material is preferably 2:1. The molar ratio of the HOBT to the glutamic acid preparation raw material is preferably 1:1, and the molar ratio of the EDC to the glutamic acid preparation raw material is preferably 1:1. The fourth organic solvent is preferably anhydrous dichloromethane or anhydrous DMF. The activation is preferably performed in a protective gas atmosphere, preferably nitrogen. The activation is preferably performed in an ice bath, and the activation time is preferably 30 minutes.

[0078] After obtaining the activation solution, the present invention combines the activation solution, the glutamic acid preparation raw material, and a fifth organic solvent for a condensation reaction to obtain the glutamic acid-modified temozolomide. In the present invention, the fifth organic solvent is preferably anhydrous dichloromethane. The present invention preferably dissolves the glutamic acid preparation raw material in the fifth organic solvent to obtain a glutamic acid preparation raw material solution, and then mixes the activation solution and the glutamic acid preparation raw material solution. The condensation reaction is preferably carried out at room temperature. The condensation reaction time is preferably 15 hours. The formation of the product during the condensation reaction is preferably detected by TLC. After the condensation reaction is completed, the present invention preferably removes the organic solvent from the resulting condensation reaction solution to obtain a mixture; the mixture is redissolved in chloroform and extracted sequentially with a saturated aqueous bicarbonate solution, hydrochloric acid, and a saturated aqueous sodium chloride solution to obtain an extracted organic phase; the extracted organic phase is concentrated and separated by column chromatography to obtain the glutamic acid-modified temozolomide. The method for removing the organic solvent is preferably rotary evaporation. The molar concentration of the hydrochloric acid is preferably 1 mol / L. The concentration is preferably rotary evaporation. The mobile phase used in the column chromatography separation is preferably a mixed solvent of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol in the mixed solvent of dichloromethane and methanol is preferably 20:1.

[0079] In the present invention, the elemene preferably includes one or more of α-elemene, β-elemene, γ-elemene, and δ-elemene. Elemene (EIE) is a natural sesquiterpenoid compound with broad anti-tumor activity. It inhibits tumor growth and metastasis by inducing tumor cell apoptosis, inhibiting angiogenesis, and disrupting the cell cycle.

[0080] In the present invention, the liposome preparation preferably includes the following raw materials in parts by weight:

[0081] 2-8 parts of indole-oleamine, 20-40 parts of DSPE-PEG-glutathione, 20-200 parts of glutamic acid-modified temozolomide, 10-40 parts of elemene, 80-120 parts of phospholipids and 2-6 parts of cholesterol.

[0082] The raw materials for preparing the liposome preparation provided by the present invention include 2 to 8 parts by mass, preferably 3 to 7 parts, more preferably 4 to 6 parts, and in an embodiment, 5.4 parts of indole-oleamine. The indole-oleamine is prepared by condensation reaction of oleylamine and indole-2-carboxylic acid.

[0083] Based on the mass fraction of the indole-oleylamine, the raw materials for preparing the liposome preparation provided by the present invention include 20 to 40 parts of DSPE-PEG-glutathione, preferably 25 to 35 parts, and in the embodiment, it can be 31.4 parts. In the present invention, the weight-average molecular weight of PEG in the DSPE-PEG-glutathione is preferably 1000 to 10000 Da. In the present invention, the weight-average molecular weight of PEG in the DSPE-PEG-glutathione is the weight-average molecular weight of PEG used when preparing the DSPE-PEG-glutathione.

[0084] In the present invention, the DSPE-PEG-glutathione is preferably synthesized by a Michael type addition reaction. In a specific embodiment of the present invention, the synthesis route of the DSPE-PEG-glutathione is as follows Figure 11 The DSPE-PEG-glutathione was prepared according to the method disclosed in “Optimisation of glutathione conjugation to liposomes quantified with a validated HPLC assay” (Joy N. Reginald-Opara, Darren Svirskis, Simon J. O'Carroll, Sreevalsan Sreebhavan, Justin M. Dean, Zimei Wu, International Journal of Pharmaceutics 567 (2019) 118451).

[0085] Based on the mass fraction of the indole-oleylamine, the raw materials for preparing the liposome preparation provided by the present invention include 80-120 parts of phospholipids, preferably 90-100 parts, more preferably 95-105 parts, and in the embodiment, 100 parts. In the present invention, the phospholipids preferably include one or more of soybean lecithin, egg yolk lecithin, hydrogenated lecithin, and synthetic lecithin. The hydrogenated lecithin preferably includes hydrogenated soybean lecithin and / or hydrogenated egg yolk lecithin. The synthetic lecithin preferably includes synthetic lecithin. The synthetic lecithin is a phospholipid synthesized by a chemical method.

[0086] Based on the mass fraction of the indole-oleylamine, the raw materials for preparing the liposome preparation provided by the present invention include 2 to 6 parts of cholesterol, preferably 3 to 5 parts, and in the embodiment, 4 parts.

[0087] Based on the mass fraction of the indole-oleylamine, the raw materials for preparing the liposome preparation provided by the present invention include 20 to 200 parts of glutamic acid-modified temozolomide, preferably 21 to 190 parts, more preferably 22 to 190 parts, and in the embodiment, it can be 23.3 parts, 87.8 parts, or 186.5 parts.

[0088] Based on the mass fraction of the indole-oleylamine, the raw materials for preparing the liposome preparation provided by the present invention include 10 to 40 parts of elemene, preferably 12 to 38 parts, and more preferably 15 to 36 parts. In the embodiment, it can be 15.6 parts or 35.2 parts.

[0089] The present invention provides a method for preparing the liposome preparation described in the above technical solution, comprising the following steps:

[0090] dissolving the raw materials for preparing the liposome preparation in a sixth organic solvent to obtain a drug mixed solution;

[0091] The organic solvent is removed from the drug mixed solution, and then hydrated with a hydration medium to obtain the liposome preparation.

[0092] In the present invention, the raw materials for preparing the liposome preparation are dissolved in a sixth organic solvent to obtain a drug mixture solution. In the present invention, the sixth organic solvent preferably comprises one or more of chloroform, ethanol, dichloromethane, and methanol. The ratio of indole-oleylamine to the third organic solvent is preferably (2-8) mg:100 mL. The total mass of the raw materials for preparing the liposome preparation and the amount of the sixth organic solvent are preferably (1-3) mg:1 mL.

[0093] After obtaining the drug mixture solution, the present invention removes the organic solvent from the drug mixture solution and then hydrates it with a hydration medium to obtain the liposome preparation. In the present invention, the method for removing the organic solvent is preferably rotary evaporation. After removing the organic solvent from the drug mixture solution, a drug lipid film is obtained. In the present invention, the drug lipid film and the hydration medium are preferably mixed for hydration. The hydration medium preferably comprises one or more of water, physiological saline, and a phosphate buffer solution with a pH of 7.4. The water is preferably deionized water. The hydration is preferably performed under stirring, at a temperature of 45-50°C, and for a time of 0.5-1 hour. After completion of the hydration, a hydrated solution is obtained. The present invention also preferably includes ultrasonically dispersing the hydrated solution and then filtering it to obtain the liposomes. The ultrasonic dispersion is preferably performed using a probe sonicator. The ultrasonic dispersion conditions preferably include: an ultrasonication time of 20-40 minutes, an ultrasonication period of 5 seconds, an ultrasonication rest period of 5 seconds, an ultrasonication temperature of ≤70°C, and an ultrasonication power of 225-300 W. The filtration preferably includes filtering using a 0.45 μm water filter membrane and a 0.22 μm water filter membrane in sequence.

[0094] The present invention provides the use of the liposome preparation described in the above technical solution or the liposome preparation prepared by the preparation method described in the above technical solution in the preparation of brain-targeted anticancer drugs.

[0095] In the present invention, the brain-targeted anticancer drug preferably includes a targeted drug for preparing brain glioma.

[0096] In summary, the present invention utilizes indole residues and glutathione groups to modify lipid membranes. The indole residues and glutathione groups have a targeting effect or the function of promoting transmembrane delivery. At the same time, the present invention optimizes the types of raw materials for liposome preparation, thereby enhancing the liposome's targeting ability to gliomas, thereby improving the efficiency of the loaded drug penetrating the blood-brain barrier.

[0097] In order to further illustrate the present invention, 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 scope of protection of the present invention.

[0098] The specific descriptions of the liposome abbreviations used in the following examples and the drawings are shown in Table 1.

[0099] Table 1 Specific description of the liposome abbreviations used in the examples and figures of the specification

[0100]

[0101] according to Figure 12 The synthetic route of the invention is as follows:

[0102] Oleylamine and indole-2-carboxylic acid were used as starting materials, with a molar equivalent ratio of 1:1.5. First, indole-2-carboxylic acid, HOBT, and EDC (equimolar equivalents of HOBT, EDC, and oleylamine) were dissolved in anhydrous dichloromethane and activated under a nitrogen atmosphere in an ice bath for 30 minutes. Oleylamine, dissolved in anhydrous dichloromethane, was then added to the solution. The reaction was continued at room temperature for 48 hours, and product formation was monitored by TLC. After completion of the reaction, the dichloromethane was removed by rotary evaporation, and the mixture was redissolved in chloroform. Extraction was performed sequentially with 5 wt% NaHCO₃ solution and saturated NaCl. The organic phase was concentrated by rotary evaporation and separated by column chromatography (mobile phase: n-hexane:ethyl acetate = 4:1) to obtain the indole-oleylamine molecule.

[0103] The preparation method of the glutamic acid-modified temozolomide prodrug molecule in the following examples is as follows:

[0104] The method uses glutamic acid with an amino group exposed and a carboxyl group protected, a second-generation dendritic glutamic acid or a third-generation dendritic glutamic acid and carboxylated temozolomide (TMZ-COOH) as raw materials, and the reaction is carried out with the molar equivalent ratio of the carboxyl group of the carboxylated temozolomide to the amino group of the glutamic acid raw material (glutamic acid with an amino group exposed and a carboxyl group protected, a second-generation dendritic glutamic acid or a third-generation dendritic glutamic acid) being 2:1. First, TMZ-COOH, HOBT, and EDC (equimolar equivalents of HOBT, EDC and amine-exposed, carboxyl-protected glutamic acid, second-generation dendritic glutamic acid, or third-generation dendritic glutamic acid) were dissolved in a mixed solvent of anhydrous dichloromethane and anhydrous DMF and activated under a nitrogen atmosphere in an ice bath for 30 min. Then, amine-exposed, carboxyl-protected glutamic acid, second-generation dendritic glutamic acid, or third-generation dendritic glutamic acid dissolved in anhydrous dichloromethane were added to the above solution and reacted at room temperature for 15 h. The formation of the product was monitored by TLC. After the reaction, the solvent was removed by rotary evaporation, and the mixture was redissolved in chloroform and extracted with saturated NaHCO3, 1 mol / L HCl, and saturated NaCl solutions. The mixture was concentrated by rotary evaporation and separated by column chromatography (mobile phase V dichloromethane:V methanol = 20:1) to obtain the glutamic acid-modified temozolomide prodrug molecule.

[0105] Example 1

[0106] This embodiment provides a brain-targeted liposome preparation modified with indole and glutathione and loaded with elemene and temozolomide prodrug molecules. The preparation method specifically includes:

[0107] Step 1: Prepare solution A, which comprises: 0.54 mg of indole-oleamine, 3.14 mg of DSPE-PEG-glutathione, 2.33 mg of second-generation dendrimer glutamic acid-modified temozolomide (i.e., second-generation dendrimer glutamic acid-temozolomide), 1.56 mg of β-elemene, 10 mg of soy lecithin, and 0.4 mg of cholesterol. Add the above components to chloroform to obtain solution A.

[0108] Step 2: Solution A was rotary evaporated to dryness to form a lipid film; pH 7.4 phosphate buffer was added to the container containing solution A, and the mixture was rotated and hydrated in a 60°C water bath for 0.5-1 h. Ultrasonic dispersion was performed using the following probe ultrasound parameters: ultrasound time 30 min, ultrasound 5 s on, 5 s off, upper temperature 70°C, and ultrasound power 225 W. The mixture was filtered through 0.45 μm and 0.22 μm aqueous filter membranes in sequence to obtain indole- and glutathione-modified brain-targeted liposomes loaded with elemene and temozolomide prodrug molecules (abbreviated as PCIGT@β-ELE).

[0109] Comparative Example 1

[0110] The difference from Example 1 is that β-elemene is not contained, and indole- and glutathione-modified brain-targeted liposomes loaded with temozolomide prodrug molecules (abbreviated as PCIGT) are prepared.

[0111] Comparative Example 2

[0112] The difference from Example 1 is that DSPE-PEG-glutathione is not included, and indole-modified liposomes loaded with elemene and temozolomide prodrug molecules (abbreviated as PCIT@β-ELE) are prepared.

[0113] Comparative Example 3

[0114] The difference from Example 1 is that indole-oleamine is not contained, and glutathione-modified liposomes loaded with elemene and temozolomide prodrug molecules (abbreviated as PCGT@β-ELE) are prepared.

[0115] Comparative Example 4

[0116] The difference from Example 1 is that indole-oleylamine and DSPE-PEG-glutathione are not included, and liposomes loaded with elemene and temozolomide prodrug molecules (abbreviated as PCT@β-ELE) are prepared.

[0117] Example 2

[0118] This embodiment provides a brain-targeted liposome preparation modified with indole and glutathione and loaded with elemene and temozolomide prodrug molecules. The preparation method specifically includes:

[0119] Step 1: Prepare solution A, which comprises: 0.54 mg of indole-oleamine, 3.14 mg of DSPE-PEG-glutathione, 8.78 mg of third-generation dendritic glutamic acid-modified temozolomide (i.e., third-generation dendritic glutamic acid-temozolomide), 1.56 mg of β-elemene, 10 mg of soy lecithin, and 0.4 mg of cholesterol. Add the above components to chloroform to obtain solution A.

[0120] Step 2: Solution A was evaporated to dryness by rotary evaporation to form a lipid film; phosphate buffer (pH = 7.4) was added to the container containing solution A, and the mixture was hydrated in a 50°C water bath for 0.5-1 h. Ultrasonic dispersion was performed using the following probe ultrasound parameters: ultrasound time 30 min, ultrasound 5 s on, 5 s off, upper temperature 70°C, and ultrasound power 225 W. The mixture was filtered through 0.45 μm and 0.22 μm aqueous filter membranes in sequence to obtain indole- and glutathione-modified brain-targeted liposomes loaded with elemene and temozolomide prodrug molecules.

[0121] Example 3

[0122] The difference from Example 2 is that the amount of β-elemene is 3.52 mg, and the amount of the third-generation dendritic glutamic acid-modified temozolomide is 18.65 mg.

[0123] Example 4

[0124] The difference from Example 2 is that α-elemene is used.

[0125] Example 5

[0126] The difference from Example 2 is that ethanol is used as the solvent for preparing solution A.

[0127] Example 6

[0128] The difference from Example 2 is that deionized water is used as the hydration medium.

[0129] Results Test

[0130] Laser dynamic light scattering was used to measure the particle size, distribution and ζ-potential of the liposome preparations prepared in each example and comparative example. The results are shown in Table 2.

[0131] Table 2 Particle size and distribution of brain-targeted liposomes and ζ-potential results in Examples and Comparative Examples

[0132]

[0133] Figure 1 The particle size distribution of the liposome preparations prepared in Example 1 and Comparative Example 1 is shown in Table 2 and Figure 1It can be seen that the particle size of the liposome preparations prepared by the present invention is less than 200 nm and the particle size distribution is uniform. The zeta potential of the liposome preparations prepared by the present invention is negative.

[0134] Example 7: Hemolytic activity evaluation

[0135] Fresh whole blood was collected from C57 mice and placed in sodium heparinized blood collection tubes. The tubes were then centrifuged at 1500 rpm for 5-10 minutes. The supernatant was removed from the tubes, and PBS buffer was added to wash the red blood cells. The tubes were centrifuged again until the supernatant was clear. Finally, the red blood cells were resuspended in PBS buffer to a 2% (w / v) solution. The liposome preparation prepared as described in Example 1 was diluted in PBS buffer to concentrations of 25, 50, 100, 200, and 400 μg / mL. 0.2 mL of each liposome preparation at different concentrations was mixed with an equal volume of 2% red blood cell suspension. The mixtures were incubated at 37°C on a constant temperature shaker for 1 hour and centrifuged at 10,000 rpm for 10 minutes. The supernatant was then collected and the absorbance (A) was measured at 540 nm using a microplate reader. The result of co-incubation of 1% Triton (polyethylene glycol octylphenyl ether, Triton X-100) with erythrocytes was used as a positive control, i.e., the hemolysis rate was 100%; the result of co-incubation of PBS with erythrocytes was used as a negative control, i.e., the hemolysis rate was 0%.

[0136] The formula for calculating the hemolysis rate of each group of liposomes is: Hemolysis rate = (A 样品 -A 阴性对照 ) / (A 阳性对照 -A 阴性对照 )×100%.

[0137] The results are as follows Figure 2 As shown in the data, within the concentration range of 25 to 400 μg / mL of the liposome preparation prepared in Example 1, the liposome preparation did not cause significant hemoglobin release. When the concentration of the liposome preparation was 400 μg / mL, the hemolysis rate was only 5.23±0.31%, indicating that the liposome preparation prepared in Example 1 had good blood compatibility.

[0138] Example 8: Anti-nonspecific protein adsorption assay

[0139] The liposome preparations prepared by the methods described in Example 1 and Comparative Example 1 were incubated with bovine serum albumin (BSA) and histone solutions in PBS (phosphate buffered saline) at pH 7.4 at 37°C and 120 rpm. The sample concentration was 0.1 mg / mL, and the BSA and histone concentrations were 0.1 mg / mL. After 2 hours, the mixture was centrifuged at 10,000 g for 15 minutes to precipitate the BSA and histone adsorbed by the material. The concentrations of BSA and histone in the supernatant were determined using a UV-visible-near-infrared spectrophotometer, with their maximum absorbances at 280 nm and 275 nm. The adsorption of BSA and histone on the material sample was then calculated using standard calibration curves for BSA and histone. Three measurements were performed for each sample, and the results were averaged.

[0140] The results are as follows Figure 3 As shown in the figure, the adsorption capacity of the liposome preparation prepared by the method described in Example 1 for BSA is 17.25±7.06%, and the adsorption capacity for Histone is 20.05±9.05%. This is because the isoelectric point of BSA is 5.4, and the isoelectric point of Histone is 10.8. Under the solution condition of pH=7.4, BSA is negatively charged and Histone is positively charged. Therefore, the liposome preparation with a negative ζ-potential has a lower adsorption capacity for BSA, and the adsorption capacity for both proteins is less than 21%, indicating that the material has good anti-protein adsorption performance.

[0141] Example 9: Cytotoxicity Experiment

[0142] The cytotoxicity of brain-targeted liposomes to U-87 MG (human astrocytic glioblastoma cells) and GL261 (mouse glioma cells) was investigated by MTT assay. 3Cells were seeded into 96-well plates at a concentration of 100 μg / well and incubated in a cell culture incubator at 37°C and 5% CO2 for 24 h. The liposome preparations (PCIGT@β-ELE, PCIT@β-ELE, PCGT@β-ELE, and PCT@β-ELE) prepared in Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4 were gradually diluted with DMEM high-glucose medium containing 10% fetal bovine serum (FBS) to produce solutions with β-elemene concentrations of 6.25, 12.5, 25, 50, 100, and 200 μg / mL. Similarly, β-elemene (β-ELE) alone, temozolomide (TMZ) alone, a second-generation dendritic glutamic acid-modified temozolomide prodrug (TMZ-G2E), and a mixture of a second-generation dendritic glutamic acid-modified temozolomide prodrug and β-elemene (β-ELE / TMZ-G2E mix) were administered. In the β-ELE / TMZ-G2E mix, β-ELE and TMZ-G2E were combined at a 3:1 molar ratio. All cells were diluted with DMEM complete medium to the same concentrations (6.25, 12.5, 25, 50, 100, and 200 μg / mL) and incubated in cell culture plates for 48 hours. The medium was discarded, and 100 μL of PBS solution containing 0.5 mg / mL MTT was added to the 96-well plate and incubated in a cell culture incubator for another 4 hours. Discard the culture medium and add DMSO (100 μL) to the cell wells. Place the wells in a microplate reader and shake evenly. Measure the absorbance at 570 nm. 样品 The OD value of the cell wells without drug treatment was used as the blank group, and the OD value of the culture medium group without cell solution and drug was used as the background group. The formula for calculating the cell survival rate of the drug group is: survival rate = (OD 样品 -OD 背景 ) / (OD 空白 -OD 背景 )×100%.

[0143] The results are as follows Figure 4 As shown, the IC values of the temozolomide alone group and the β-elemene alone group on U-87 MG cells were 50 The values were 102.54 μg / mL and 115.42 μg / mL, respectively. The IC values of the brain-targeted liposomes prepared in Example 1 for U-87 MG cells were 50 The value was 50.02 μg / mL, which was only 0.49 and 0.43 times of the groups treated with temozolomide and β-elemene alone, respectively; the IC 50The values were 108.04 μg / mL and 89.47 μg / mL, respectively. The IC values of the brain-targeted liposomes prepared in Example 1 on GL261 cells were 50 The value was 53.25 μg / mL, which was only 0.49 and 0.60 times that of the groups administered with temozolomide and β-elemene alone, respectively. The results showed that the brain-targeted liposomes prepared in Example 1 had significant inhibitory effects on both U-87 MG cells and GL261 cells. With the increase of the dosage, the inhibitory effect on tumor cells gradually increased, significantly reducing the IC value of the group administered with temozolomide alone. 50 The results showed that temozolomide and β-elemene had a coordinated antitumor effect.

[0144] Example 10: Cellular uptake experiment

[0145] According to the preparation method of the liposome preparation in Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4, elemene was replaced with doxorubicin (DOX) with red fluorescence to achieve intracellular tracing of the liposome preparation. U-87 MG cells and GL261 cells were cultured at 2×10 4 Each dish was inoculated into a glass-bottomed dish and cultured at 37°C, 5% CO2 for 24 h. The old culture medium was discarded, and the above four DOX liposome preparations and DOX were diluted with DMEM complete medium and added to the glass-bottomed dish. The DOX concentration was 10 μg / mL. The cells were incubated in a cell culture incubator for 2 or 4 h. Subsequently, the drug-containing culture medium was discarded, and Hoechst 33342 diluted in DMEM complete medium in advance was added to a final concentration of 10 μg / mL. The cells were incubated in an incubator for 20 min, the old culture medium was discarded, and the cells were washed three times with PBS. The cells were then observed under a laser confocal microscope.

[0146] The results are as follows Figure 5As shown in the figure, in U-87 MG cells and GL261 cells, the red fluorescence intensity at 2 h was greater than that at 4 h for each control group. This is because the liposome preparation had not been fully internalized by the cells at 2 h. A certain fluorescence intensity was observed in the DOX group, which may be because free DOX is a small molecule that can passively diffuse through the cell membrane into the cell. Only weak fluorescence was observed in the DOX-loaded liposome preparation group (PCT@DOX), which may be because the ζ-potential of the PCT liposomes prepared by the method of Comparative Example 4 is negative, which repels the negative charge of the cell membrane. Stronger fluorescence was observed in the DOX-loaded indole-modified liposome preparation group (PCIT@DOX) and the DOX-loaded glutathione-modified liposome preparation group (PCGT@DOX), which may be because the PCIT@DOX liposome preparation can destroy the membrane structure of the cell membrane due to the presence of indole residues. The presence of glutathione groups in the PCGT@DOX liposome formulation allows for better entry into tumor cells via the glutathione transporter. The brain-targeted liposome formulation (PCIGT@DOX) loaded with DOX and modified with indole and glutathione exhibited the strongest fluorescence, due to the synergistic effect of indole and glutathione, which enhances its transcellular transmembrane entry. These results demonstrate that the brain-targeted liposomes prepared by the method of Example 1 possess excellent glioma-targeting capabilities.

[0147] Example 11: Lysosomal escape experiment

[0148] According to the preparation method of brain-targeted liposome preparation in Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4, elemene was replaced with DOX to prepare DOX-labeled brain-targeted liposomes. U-87 MG cells and GL261 cells were cultured at 2×10 4 100 cells / dish were inoculated into glass-bottom dishes and cultured at 37°C, 5% CO2 for 24 h. The old culture medium was discarded, and the above four DOX liposome preparations and DOX were diluted with DMEM complete medium and added to the dishes. The DOX concentration was 10 μg / mL. The dishes were incubated in a cell culture incubator for 2 h or 4 h. The drug-containing culture medium was discarded, and Hoechst 33342 and Lyso-Tracker Green diluted in DMEM complete medium in advance were added. The final concentrations of Hoechst 33342 and Lyso-Tracker Green were 10 μg / mL and 50 nM, respectively. The cells were incubated in an incubator for 20 min, the old culture medium was discarded, and the cells were washed three times with PBS. The cells were then observed under a laser confocal microscope.

[0149] The results are as follows Figure 6As shown, in U-87 MG cells and GL261 cells, the green fluorescence intensity in the DOX group did not change much after 2 h and 4 h of culture, indicating that free DOX had difficulty escaping from lysosomes. The PCT@DOX group had difficulty entering the cell, so only weak green fluorescence was observed. Extremely low green fluorescence was observed in the PCIT@DOX and PCIGT@DOX groups, which is because the indole residues inserted into the lysosomal lipid bilayer structure after entering the lysosome, destroying the lysosome. PCGT@DOX also observed low green fluorescence, which may be because the basic structure of liposomes is similar to that of biological membranes, allowing lysosomal escape through membrane fusion. These results indicate that the brain-targeted liposomes prepared by the method of Example 1 have excellent lysosomal escape properties.

[0150] Example 12: Brain endothelial cell transport performance experiment

[0151] The ability of brain-targeted liposomes to cross the blood-brain barrier and enter the brain tissue was evaluated by endocytosis experiments of mouse brain endothelial cells (bEnd.3). According to the preparation methods of brain-targeted liposome preparations in Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4, elemene was replaced with DOX to prepare DOX-labeled brain-targeted liposome preparations. bEnd.3 cells were cultured at 2×10 4 100 cells / dish were inoculated into glass-bottom dishes and cultured at 37°C, 5% CO2 for 24 h. The old culture medium was discarded, and the above four DOX liposome preparations and pure DOX were diluted with DMEM complete medium and added to the glass-bottom dishes. The DOX concentration was 10 μg / mL. The cells were incubated in a cell culture incubator for 2 h or 4 h. The drug-containing medium was discarded, and Hoechst 33342 and Lyso-Tracker Green, which had been diluted in DMEM complete medium in advance, were added. The final concentration of Hoechst 33342 was 10 μg / mL and the final concentration of Lyso-Tracker Green was 50 nM. The cells were incubated in an incubator for 20 min. The old culture medium was discarded, and the cells were washed three times with PBS. The cells were observed under a laser confocal microscope.

[0152] The results are as follows Figure 7As shown, in bEnd.3 cells, the red fluorescence intensity at 2 h was greater than that at 4 h for each control group. This is because the liposome formulation had not yet been fully internalized by the endothelial cells at 2 h. Because indole residues and glutathione groups can target amino acid receptors and glutathione transporters, respectively, which are highly expressed in brain endothelial cells, both the PCIT@DOX and PCGT@DOX groups showed stronger fluorescence than the DOX group alone, demonstrating that liposome formulations modified with indole residues and glutathione groups can help small molecules more efficiently cross the blood-brain barrier through endothelial cell transport. Among them, the strongest red fluorescence was observed in the PCIGT@DOX group, because the presence of the dual targeting groups further enhanced the internalization of brain-targeted liposomes by endothelial cells. These results show that the brain-targeted liposome formulation prepared in Example 1 has excellent blood-brain barrier penetration ability.

[0153] Example 13: Establishing an in vitro blood-brain barrier model to evaluate the ability of liposomes to penetrate the blood-brain barrier

[0154] According to the preparation method of brain-targeted liposome preparation in Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4, elemene was replaced with DOX to prepare DOX-labeled brain-targeted liposomes. 4 bEnd.3 cells were prepared into 200 μL of culture medium suspension and injected into the upper chamber of 24-well Transwell for monolayer culture. 5 GL261 cells were seeded in 800 μL of culture medium and injected into the lower chamber of a Transwell. When the monolayer's TEER reached 200 Ω·cm² or higher, the blood-brain barrier model was ready for further experiments. The four DOX liposome formulations and DOX alone were diluted in DMEM complete medium and added to the dish to a DOX concentration of 20 μg / mL. The cells were incubated in a cell culture incubator for 4 hours. The drug-containing medium was discarded, and Hoechst 33342, previously diluted in DMEM complete medium, was added to a final concentration of 10 μg / mL. The remaining medium was discarded, and the cells were washed three times with PBS before observation under an inverted fluorescence microscope.

[0155] The results are as follows Figure 8As shown, the present invention observed that the PCIT@DOX group exhibited very weak red fluorescence (from DOX), which may be due to the consumption of a large amount of indole residues when penetrating the bEnd.3 tight junction. The PCGT@DOX group and the PCIGT@DOX group, with the same glutathione molar equivalent, showed stronger red fluorescence in the latter, further demonstrating that indole residues can disrupt membrane structure and enhance lysosomal escape. The results show that the brain-targeted liposome preparation prepared in Example 1 can cross the in vitro blood-brain barrier model and enter glioma cells.

[0156] Example 14: In vivo biodistribution experiment

[0157] According to the preparation method of brain-targeted liposomes in Example 1 and Comparative Example 4, elemene was replaced with a near-infrared fluorescent dye Dir (excitation wavelength of about 754 nm, emission wavelength of about 778 nm) which is convenient for in vivo tracing, to prepare Dir-labeled brain-targeted liposomes. The above-mentioned Dir liposomes (Dir concentration of 0.2 mg / kg) were injected into C57BL / 6 mice bearing brain glioma via tail vein injection, and then in vivo imaging was performed at predetermined time points after injection (1, 2, 3, 4, 6, 8, 10, 24, and 48 hours). Finally, the heart, lung, liver, kidney, spleen, and brain were collected and imaged in vitro 48 hours after injection.

[0158] The results are as follows Figure 9 As shown, mice with gliomas injected with PCIGT@Dir exhibited stronger fluorescence in the brain than those with the PCT@Dir group. Furthermore, some intracranial enrichment of PCIGT@Dir was observed within the mouse skull 1 hour after injection, reaching a maximum at 24 hours and maintaining the effect for 48 hours. The in vitro distribution results were consistent with the in vivo distribution. These results demonstrate that the brain-targeted liposome formulation prepared in Example 1 can effectively cross the blood-brain barrier and accumulate continuously in brain tissue.

[0159] Example 15: In vivo anti-glioma experiment

[0160] A C57BL / 6 mouse model bearing brain glioma was constructed, and the in vivo anti-glioma performance of the indole- and glutathione-modified brain-targeted liposome formulation loaded with elemene and temozolomide prodrug prepared in Example 1 was evaluated. 18 tumor-bearing C57BL / 6 mice were randomly divided into three groups, with 6 mice in each group. They were (1) saline control group (abbreviated as 0.9% NaCl group); (2) β-elemene injection group (abbreviated as β-ELE group); and (3) PCIGT@β-ELE group. The drug was administered by tail vein injection on days 1, 3, 5, 7, 9, 11, and 13, for a total of 7 treatments, with 20 mg / kg β-ELE administered each time. After different treatment time points, the tumor-bearing mice underwent bioluminescence imaging on days 0, 5, 10, and 15 to evaluate tumor inhibition.

[0161] The results are as follows Figure 10 As shown, after 15 days of treatment, the tumor fluorescence intensity in the PCIGT@β-ELE group was minimal compared to the other groups. The β-ELE group also had a modest inhibitory effect on gliomas, but the inhibitory effect was relatively weak. These results demonstrate that the indole- and glutathione-modified brain-targeted liposome formulation loaded with elemene and a glutamic acid-modified temozolomide prodrug exhibits potent anti-glioma efficacy.

[0162] As can be seen from the above examples, the indole and glutathione-modified brain-targeted liposome preparation loaded with elemene and temozolomide prodrug provided by the present invention has excellent transmembrane transport ability, can efficiently penetrate the blood-brain barrier and be taken up by brain glioma cells, and at the same time exhibits excellent endosomal escape properties. In addition, the synergistic effect of glutamic acid-modified temozolomide and elemene can effectively reduce the toxic side effects of temozolomide and elemene, and reduce the IC 50 , and also enhanced the anti-tumor effect. This liposomal formulation is expected to be a highly effective and low-toxic glioma treatment method, providing a new solution to overcome the key challenges in brain tumor treatment.

[0163] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A liposome, characterized in that The liposome membrane is modified with glutathione groups and indole residues; the liposome comprises the following raw materials in parts by weight: 2-8 parts of indole-oleamine, 20-40 parts of DSPE-PEG-glutathione, 80-120 parts of phospholipids, and 2-6 parts of cholesterol; The chemical structure of the indole-oleylamine is as follows: ； The chemical structural formula of the DSPE-PEG-glutathione is as follows: 。 2. The liposome according to claim 1, wherein The phospholipids include one or more of soybean lecithin, egg yolk lecithin, hydrogenated lecithin and synthetic lecithin.

3. Use of the liposome according to claim 1 or 2 in the preparation of a brain-targeted liposome preparation.

4. A liposome preparation, characterized in that The liposome preparation comprises the following raw materials in parts by weight: 2-8 parts of indole-oleamine, 20-40 parts of DSPE-PEG-glutathione, 20-200 parts of glutamic acid-modified temozolomide, 10-40 parts of elemene, 80-120 parts of phospholipids, and 2-6 parts of cholesterol; The chemical structure of the indole-oleylamine is as follows: ； The chemical structural formula of the DSPE-PEG-glutathione is as follows: ； The glutamic acid-modified temozolomide is one or more of the following chemical structures: 。 5. The liposome preparation according to claim 4, characterized in that The elemene includes one or more of α-elemene, β-elemene, γ-elemene and δ-elemene.

6. The method for preparing the liposome preparation according to claim 4 or 5, characterized in that: The following steps are involved: dissolving the raw materials for preparing the liposome preparation in an organic solvent to obtain a drug mixed solution; The organic solvent is removed from the drug mixed solution, and then hydrated with a hydration medium to obtain the liposome preparation.

7. Use of the liposome preparation according to any one of claims 4 or 5 or the liposome preparation prepared by the preparation method according to claim 6 in the preparation of brain-targeted anticancer drugs.

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