Liposome and application thereof, liposome preparation and preparation method and application thereof

Abstract

The invention belongs to the technical field of liposome pharmaceutical preparations, and particularly relates to a liposome and application thereof, a liposome preparation and a preparation method and application thereof. The invention provides a liposome. A glutathione group and an indole residue are modified on a liposome membrane. The lipidosome provided by the invention has excellent transmembrane transport capacity, can efficiently penetrate through a blood-brain barrier and can be taken by glioma cells, and also shows excellent endosome escape characteristic, so that the technical problem of low blood-brain barrier transmittance in the glioma treatment process of temozolomide is effectively solved. Meanwhile, the invention provides a liposome preparation, the delivery efficiency and the anti-tumor curative effect are improved, the liposome preparation has an inhibiting effect on human / mouse glioma cells, the wide clinical application potential is shown, and an efficient and low-toxicity novel treatment scheme is provided for glioma patients.

Description

Technical Field

[0001] The invention belongs to the technical field of liposome pharmaceutical preparations, and specifically 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 in the central nervous system, which is highly invasive and heterogeneous. Due to its rapid proliferation, diffuse growth, and unclear boundaries with normal brain tissue, it is difficult to treat. Traditional treatments, such as surgery, radiotherapy, and chemotherapy, are often limited in effectiveness due to the limitations of the blood-brain barrier (BBB). The existence of the blood-brain barrier makes it difficult for many chemotherapy drugs to effectively reach the tumor area, thereby limiting the effectiveness of treatment.

[0003] Temozolomide (TMZ), as a standard chemotherapy drug for the treatment of glioma, exerts its anti-tumor effect mainly by alkylating DNA. Although temozolomide can prolong the survival of some patients, its efficacy is limited by the low permeability of the blood-brain barrier, which reduces its clinical effect. Summary of the invention

[0004] The purpose of the present invention is 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 characteristics, thereby effectively solving the technical problem of low blood-brain barrier permeability during temozolomide treatment of glioma.

[0005] In order to achieve the above object, the present invention provides the following technical solutions: The invention provides a liposome, wherein the liposome membrane is modified with glutathione groups and indole residues.

[0006] Preferably, the liposome comprises the following raw materials in parts by weight: 2-8 parts of indole-oleylamine, 20-40 parts of DSPE-PEG-glutathione, 80-120 parts of phospholipids and 2-6 parts of cholesterol; The indole-oleylamine is prepared by condensing oleylamine and indole-2-carboxylic acid.

[0007] Preferably, the phospholipids include one or more of soybean lecithin, egg yolk lecithin, hydrogenated lecithin and synthetic lecithin; The raw material for preparing the DSPE-PEG-glutathione includes polyethylene glycol, and the weight average molecular weight of the polyethylene glycol is 1000-10000 Da.

[0008] The present invention provides the use of the liposome described in the above technical solution in the preparation of a brain-targeted liposome preparation.

[0009] The present invention provides a liposome preparation, comprising the liposome described in the above technical solution; Also included are drug molecules encapsulated in the vesicles of the liposomes, wherein the drug molecules include glutamic acid-modified temozolomide and elemene.

[0010] Preferably, the glutamic acid-modified temozolomide comprises one or more of the following chemical structures: .

[0011] Preferably, the elemene includes one or more of α-elemene, β-elemene, γ-elemene and δ-elemene.

[0012] Preferably, the liposome preparation comprises the following raw materials in parts by weight: 2-8 parts of indole-oleylamine, 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.

[0013] The present invention provides a method for preparing the liposome preparation described in the above technical solution, comprising the following steps: 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 hydrating medium to obtain the liposome preparation.

[0014] 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.

[0015] The present invention provides a liposome, wherein the liposome membrane is modified with a glutathione group and an indole residue. The present invention uses a glutathione group and an indole residue as a dual targeting group on the liposome membrane, wherein the glutathione group can enable the liposome to better enter the tumor cell through the glutathione transporter; the indole residue can help small molecule drugs (temozolomide and elemene) to be more efficiently transported through endothelial cells, thereby penetrating the blood-brain barrier, and the indole residue can destroy the membrane structure of the tumor cell membrane, insert into the lysosomal lipid bilayer structure after entering the lysosome, destroy the lysosome, and enhance the lysosomal escape. The synergistic effect of the indole residue and the glutathione group in the present invention further enhances the endocytosis of the liposome by the endothelial cells and enhances the transmembrane endocytosis ability of the liposome. In summary, 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 characteristics, thereby effectively solving the technical problem of low blood-brain barrier permeability during the treatment of glioma with temozolomide.

[0016] The present invention provides a liposome preparation, comprising the liposome described in the above technical solution; and also comprising drug molecules encapsulated in the vesicles of the liposome, 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, and 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 human / mouse glioma cells, showing a wide range of clinical application potential, and providing glioma patients with a new, efficient and low-toxic treatment option.

[0017] 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, and 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

[0018] Figure 1 This is a particle size distribution diagram of the brain-targeted liposome preparation loaded with elemene and temozolomide prodrug molecules prepared by the present invention; 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; Figure 3To evaluate the anti-nonspecific protein adsorption of the brain-targeted liposome preparation loaded with elemene and temozolomide prodrug molecules prepared by the present invention; 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; Figure 5 The cell uptake efficiency of the brain-targeted liposome preparation loaded with elemene and temozolomide prodrug molecules prepared by the present invention; 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); 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); Figure 8 The in vitro blood-brain barrier permeability evaluation of the brain-targeted liposome preparation loaded with elemene and temozolomide prodrug molecules prepared by the present invention; Fig. 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; Fig.10 The effect of the brain-targeted liposome preparation loaded with elemene and temozolomide prodrug molecules prepared by the present invention on treating brain glioma in vivo; Fig.11 The synthetic route of DSPE-PEG-glutathione; Fig.12 This is a synthetic route for indole-oleylamine. DETAILED DESCRIPTION

[0019] The invention provides a liposome, wherein the liposome membrane is modified with glutathione groups and indole residues.

[0020] 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 in indole-oleylamine. The indole-oleylamine is prepared by condensation reaction of oleylamine and indole-2-carboxylic acid.

[0021] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0022] In the present invention, the indole residues 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).

[0023] In the present invention, the liposome preferably comprises the following preparation raw materials in parts by weight: 2-8 parts of indole-oleylamine, 20-40 parts of DSPE-PEG-glutathione, 80-120 parts of phospholipids and 2-6 parts of cholesterol; The indole-oleylamine is prepared by condensing oleylamine and indole-2-carboxylic acid.

[0024] In terms of mass fraction, the raw material for preparing the liposome provided by the present invention comprises 2 to 8 parts of indole-oleylamine, preferably 3 to 7 parts, more preferably 4 to 6 parts, and in the embodiment, 5.4 parts. The indole-oleylamine is prepared by condensation reaction of oleylamine and indole-2-carboxylic acid.

[0025] In the present invention, the preparation method of indole-oleylamine preferably comprises the following steps: 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; The activation solution, oleylamine and a second organic solvent are mixed to carry out a condensation reaction to obtain the indole-oleylamine.

[0026] The present invention mixes indole-2-carboxylic acid, HOBT, EDC and a first organic solvent for activation to obtain an activated solution. In the present invention, the molar ratio of the oleylamine to the indole-2-carboxylic acid is preferably 1.5:1. The molar ratio of the HOBT to the oleylamine is preferably 1:1, and the molar ratio of the EDC to the oleylamine is preferably 1:1. The first organic solvent is preferably anhydrous dichloromethane. The activation is carried out in a protective gas atmosphere, and the protective gas is preferably nitrogen. The activation is preferably carried out under ice bath conditions, and the activation time is preferably 30 min.

[0027] In an embodiment of the present invention, the chemical structure of the indole-oleylamine is as follows: .

[0028] After obtaining the activation solution, the present invention mixes the activation solution, oleylamine and a second organic solvent for condensation reaction to obtain 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 obtain an oleylamine solution, and then mixes the activation solution and the oleylamine solution. The condensation reaction is preferably carried out at room temperature. The time of the condensation reaction is preferably 48 h. The present invention preferably detects the formation of the product in the condensation reaction by TLC. After the condensation reaction is completed, the present invention preferably removes the organic solvent from the obtained condensation reaction liquid to obtain a mixture; the mixture is redissolved in chloroform, and extracted with a bicarbonate aqueous solution and a saturated sodium chloride aqueous solution in sequence to obtain an extracted organic phase; the extracted organic phase is concentrated and then separated by column chromatography to obtain the indole-oleylamine. The method for removing the organic solvent is preferably rotary evaporation. The mass content of the sodium bicarbonate aqueous solution is preferably 5%. The concentration is preferably rotary evaporation concentration. The mobile phase used in the column chromatography separation is a mixed solvent of n-hexane with oily fume and ethyl acetate, and the volume ratio of n-hexane to ethyl acetate in the mixed solvent of n-hexane and ethyl acetate is preferably 4:1.

[0029] Based on the mass fraction of the indole-oleylamine, the raw material for preparing the liposome provided by the present invention includes 20 to 40 parts of DSPE-PEG-glutathione, preferably 25 to 35 parts, and can be 31.4 parts in the embodiment. In the present invention, the raw material for preparing the DSPE-PEG-glutathione preferably includes phosphoethanolamine phospholipids (DSPE), polyethylene glycol (PEG) and glutathione. The weight average molecular weight of PEG used in preparing the DSPE-PEG-glutathione is preferably 1000 to 10000 Da.

[0030] 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 "Optimisation of glutathioneconjugation 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).

[0031] As a specific embodiment of the present invention, the chemical structural formula of the DSPE-PEG-glutathione is as follows: .

[0032] Based on the mass fraction of the indole-oleylamine, the raw material for preparing the liposome provided by the present invention includes 80 to 120 parts of phospholipids, preferably 90 to 100 parts, more preferably 95 to 105 parts, and in the embodiment, it can be 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.

[0033] 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.

[0034] The present invention provides a method for preparing the liposome described in the above technical solution, preferably comprising the following steps: dissolving the raw material for preparing the liposome in a third organic solvent to obtain a mixed solution; The organic solvent is removed from the mixed solution, and then hydrated with a hydration medium to obtain the liposome.

[0035] The present invention dissolves the raw materials for preparing the liposome in a third organic solvent to obtain a mixed solution. In the present invention, the third organic solvent preferably includes one or more of chloroform, ethanol, dichloromethane and methanol. The dosage ratio of the indole-oleylamine to the third organic solvent is preferably (2-8) mg: 100 mL.

[0036] After obtaining the mixed solution, the present invention removes the organic solvent from the mixed solution, and then hydrates it with a hydration medium to obtain the liposome. 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. The present invention preferably mixes the lipid film and the hydration medium for hydration. The hydration medium preferably includes one or more of water, physiological saline and a phosphate buffer with a pH value of 7.4, and the water is preferably deionized water. The hydration is preferably carried out under stirring conditions, the hydration temperature is preferably 50°C, and the time is preferably 0.5~1 h. After the hydration is completed, a hydrated solution is obtained; the present invention preferably further includes: ultrasonically dispersing the hydrated solution and then filtering it to obtain the liposome. The ultrasonic dispersion is preferably carried out using a probe ultrasonic instrument. The conditions for the ultrasonic dispersion preferably include: the ultrasonic time is preferably 20~40 min, the ultrasonic is 5 s, and the stop is 5 s, the ultrasonic temperature is ≤70°C, and the ultrasonic power is preferably 225~300 W. The filtration preferably uses a 0.45 μm water filter membrane and a 0.22 μm water filter membrane.

[0037] The present invention provides the use of the liposome 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.

[0038] The present invention provides a liposome preparation, comprising the liposome described in the above technical solution; Also included are drug molecules encapsulated in the vesicles of the liposomes, wherein the drug molecules include glutamic acid-modified temozolomide and elemene.

[0039] The liposome preparation provided by the present invention includes the liposome described in the above technical solution.

[0040] 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.

[0041] In the present invention, the glutamic acid-modified temozolomide preferably includes one or more of glutamic acid-temozolomide, second-generation dendritic glutamic acid-temozolomide and third-generation dendritic glutamic acid-temozolomide.

[0042] In the present invention, the compound structure formula of the glutamic acid-temozolomide, the second-generation dendritic glutamic acid-temozolomide, and the third-generation dendritic glutamic acid-temozolomide is as follows: .

[0043] In the present invention, the glutamic acid modified temozolomide is preferably prepared by condensation reaction of glutamic acid preparation raw material and carboxylated temozolomide (TMZ-COOH). The glutamic acid preparation raw material preferably includes glutamic acid, second-generation dendritic glutamic acid or third-generation dendritic glutamic acid with amine group exposed and carboxyl group protected. The molar ratio of the carboxyl group of the carboxylated temozolomide to the amine group of the glutamic acid preparation raw material is preferably 2:1.

[0044] In the present invention, the method for preparing the glutamic acid-modified temozolomide preferably comprises the following steps: Mixing TMZ-COOH, HOBT, EDC and a fourth organic solvent for activation to obtain an activation solution; 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.

[0045] The present invention mixes TMZ-COOH, HOBT, EDC and a fourth organic solvent for activation to obtain an activated solution. In the present invention, the molar ratio of the carboxyl group of the carboxylated temozolomide to the amine group 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 and anhydrous DMF. The activation is preferably carried out in a protective gas atmosphere, and the protective gas is preferably nitrogen. The activation is preferably carried out under ice bath conditions, and the activation time is preferably 30 min.

[0046] After obtaining the activation solution, the present invention mixes the activation solution, the glutamic acid preparation raw material and the fifth organic solvent for 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 time of the condensation reaction is preferably 15 h. The present invention preferably detects the formation of the product in the condensation reaction by TLC. After the condensation reaction is completed, the present invention preferably removes the organic solvent from the obtained condensation reaction solution to obtain a mixture; the mixture is redissolved in chloroform, and extracted with a saturated bicarbonate aqueous solution, hydrochloric acid and a saturated sodium chloride aqueous solution in sequence to obtain an extracted organic phase; the extracted organic phase is concentrated and then 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 concentration. 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.

[0047] 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 a wide range of anti-tumor activities. It inhibits tumor growth and metastasis by inducing tumor cell apoptosis, inhibiting angiogenesis and interfering with cell cycle.

[0048] In the present invention, the liposome preparation preferably includes the following preparation raw materials in parts by weight: 2-8 parts of indole-oleylamine, 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.

[0049] In terms of mass fraction, the raw materials for preparing the liposome preparation provided by the present invention include 2 to 8 parts of indole-oleylamine, preferably 3 to 7 parts, more preferably 4 to 6 parts, and in the embodiment, 5.4 parts. The indole-oleylamine is prepared by condensation reaction of oleylamine and indole-2-carboxylic acid.

[0050] 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 can be 31.4 parts in the embodiment. 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.

[0051] 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: Fig.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).

[0052] Based on the mass fraction of the indole-oleylamine, the raw materials for preparing the liposome preparation provided by the present invention include 80 to 120 parts of phospholipids, preferably 90 to 100 parts, more preferably 95 to 105 parts, and in the embodiment, it can be 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.

[0053] 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, it can be 4 parts.

[0054] 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 embodiments, it can be 23.3 parts, 87.8 parts, or 186.5 parts.

[0055] Based on the mass fraction of the indole-oleylamine, the raw material for preparing the liposome preparation provided by the present invention includes 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.

[0056] The present invention provides a method for preparing the liposome preparation described in the above technical solution, comprising the following steps: dissolving the raw materials for preparing the liposome preparation in a sixth organic solvent to obtain a drug mixed solution; The organic solvent is removed from the drug mixed solution, and then hydrated with a hydrating medium to obtain the liposome preparation.

[0057] The present invention dissolves the raw materials for preparing the liposome preparation in a sixth organic solvent to obtain a drug mixed solution. In the present invention, the sixth organic solvent preferably includes one or more of chloroform, ethanol, dichloromethane and methanol. The dosage ratio of the indole-oleylamine and 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 dosage of the sixth organic solvent are preferably (1-3) mg:1 mL.

[0058] After obtaining the drug mixed solution, the present invention removes the organic solvent from the drug mixed 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 mixed solution, a drug lipid film is obtained. The present invention preferably mixes the drug lipid film and the hydration medium for hydration. The hydration medium preferably includes one or more of water, physiological saline and a phosphate buffer with a pH value of 7.4, and the water is preferably deionized water. The hydration is preferably carried out under stirring conditions, the hydration temperature is preferably 45~50°C, and the time is preferably 0.5~1 h. After the hydration is completed, a hydrated solution is obtained; the present invention preferably further includes: ultrasonically dispersing the hydrated solution and filtering it to obtain the liposome. The ultrasonic dispersion is preferably carried out using a probe ultrasonic instrument. The conditions for the ultrasonic dispersion preferably include: the ultrasonic time is preferably 20~40 min, the ultrasonic is 5 s, and the stop is 5 s, the ultrasonic temperature is ≤70°C, and the ultrasonic power is preferably 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.

[0059] 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.

[0060] In the present invention, the brain-targeted anticancer drug preferably includes a targeted drug for preparing brain glioma.

[0061] In summary, the present invention utilizes indole residues and glutathione groups to modify lipid membranes, and 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 preparing liposomes, thereby enhancing the targeting ability of liposomes to gliomas, thereby improving the efficiency of the loaded drugs penetrating the blood-brain barrier.

[0062] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0063] The specific descriptions of the liposome abbreviations used in the following examples and the drawings of the specification are shown in Table 1.

[0064] Table 1 Specific description of the liposome abbreviations used in the examples and the accompanying drawings of the specification

[0065] according to Fig.12 The synthetic route of the invention is as follows: Oleylamine and indole-2-carboxylic acid were used as raw materials, and the molar equivalent ratio of oleylamine to indole-2-carboxylic acid was 1:1.5. First, indole-2-carboxylic acid, HOBT, and EDC (HOBT, EDC and oleylamine were in equal molar equivalents) were dissolved in anhydrous dichloromethane and activated for 30 min under nitrogen atmosphere and ice bath conditions. Then, oleylamine was dissolved in anhydrous dichloromethane and added to the above solution. The reaction was carried out at room temperature for 48 h. The formation of the product was monitored by TLC. After the reaction was completed, the dichloromethane was removed by rotary evaporation, and then the mixture was redissolved in chloroform. 5wt% NaHCO 3 The organic phase after extraction was concentrated by rotary evaporation and separated by column chromatography (the mobile phase was V n-hexane: V ethyl acetate = 4:1) to obtain the indole-oleylamine molecule.

[0066] The preparation method of the glutamic acid-modified temozolomide prodrug molecule in the following examples is as follows: The method comprises taking glutamic acid, second-generation dendritic glutamic acid or third-generation dendritic glutamic acid, and carboxylated temozolomide (TMZ-COOH) as raw materials, and reacting and feeding the materials at a molar equivalent ratio of 2:1 between the carboxyl group of carboxylated temozolomide and the amine group of the glutamic acid raw material (glutamic acid, second-generation dendritic glutamic acid or third-generation dendritic glutamic acid, which is exposed to an amine group and protected to a carboxyl group. First, TMZ-COOH, HOBT, and EDC (HOBT, EDC and amine-exposed, carboxyl-protected glutamic acid, second-generation dendritic glutamic acid or third-generation dendritic glutamic acid in equal molar equivalents) were dissolved in a mixed solvent of anhydrous dichloromethane and anhydrous DMF and activated for 30 min under nitrogen atmosphere and ice bath conditions. 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 for 15 h at room temperature. The formation of the product was monitored by TLC. After the reaction was completed, the solvent was removed by rotary evaporation, and the mixture was redissolved in chloroform and saturated with NaHCO 3 , 1 mol / L HCl and saturated NaCl solution extraction, rotary evaporation concentration, column chromatography separation (mobile phase V dichloromethane: V methanol = 20:1) to obtain glutamic acid modified temozolomide prodrug molecule.

[0067] Example 1 This embodiment provides a brain-targeted liposome preparation modified with indole and glutathione loaded with elemene and temozolomide prodrug molecules, and the preparation method specifically comprises: Step 1: Prepare solution A, which includes: 0.54 mg of indole-oleylamine, 3.14 mg of DSPE-PEG-glutathione, 2.33 mg of second-generation dendritic glutamic acid-modified temozolomide (i.e., second-generation dendritic glutamic acid-temozolomide), 1.56 mg of β-elemene, 10 mg of soybean lecithin and 0.4 mg of cholesterol, and add the above components into chloroform to obtain solution A; Step 2: The solvent of solution A was evaporated to dryness by rotary evaporation to form a lipid film; phosphate buffer with pH = 7.4 was added to the container where solution A was located, and the solution was rotated in a 60 °C water bath for hydration for 0.5-1 h, and ultrasonic dispersion was performed. The parameters of the probe ultrasonic instrument were as follows: ultrasonic time 30 min, ultrasonic 5 s on and 5 s off, upper limit temperature 70 °C, and ultrasonic power 225 W. The solution was filtered through 0.45 μm and 0.22 μm water 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).

[0068] Comparative Example 1 The difference from Example 1 is that β-elemene is not contained, and the indole and glutathione modified brain-targeted liposomes loaded with temozolomide prodrug molecules (abbreviated as PCIGT) are prepared.

[0069] Comparative Example 2 The difference from Example 1 is that DSPE-PEG-glutathione is not contained, and indole-modified liposomes loaded with elemene and temozolomide prodrug molecules (abbreviated as PCIT@β-ELE) are prepared.

[0070] Comparative Example 3 The difference from Example 1 is that indole-oleylamine is not contained, and glutathione-modified liposomes loaded with elemene and temozolomide prodrug molecules (abbreviated as PCGT@β-ELE) are prepared.

[0071] Comparative Example 4 The difference from Example 1 is that indole-oleylamine and DSPE-PEG-glutathione are not contained, and liposomes loaded with elemene and temozolomide prodrug molecules (abbreviated as PCT@β-ELE) are prepared.

[0072] Example 2 This embodiment provides a brain-targeted liposome preparation modified with indole and glutathione loaded with elemene and temozolomide prodrug molecules, and the preparation method specifically comprises: Step 1: Prepare solution A, which includes: 0.54 mg of indole-oleylamine, 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 soybean lecithin and 0.4 mg of cholesterol. The above components are added into chloroform to obtain solution A; Step 2: The solvent of solution A was evaporated to dryness by rotary evaporation to form a lipid film; phosphate buffer with pH = 7.4 was added to the container where solution A was located, and the solution was rotated in a 50 °C water bath for hydration for 0.5-1 h, and ultrasonic dispersion was performed. The parameters of the probe ultrasonic instrument were as follows: ultrasonic time 30 min, ultrasonic 5 s on and 5 s off, upper limit temperature 70 °C, and ultrasonic power 225 W. The solution was filtered through 0.45 μm and 0.22 μm water filter membranes in sequence to obtain indole and glutathione-modified brain-targeted liposomes loaded with elemene and temozolomide prodrug molecules.

[0073] Example 3 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.

[0074] Example 4 The difference from Example 2 is that α-elemene is used.

[0075] Example 5 The difference from Example 2 is that ethanol is used as the solvent for preparing solution A.

[0076] Example 6 The difference from Example 2 is that deionized water is used as the hydration medium.

[0077] Results Test Laser dynamic light scattering was used to measure the particle size, distribution and ζ-potential of the liposome preparations prepared in each embodiment and comparative example. The results are shown in Table 2.

[0078] Table 2 Particle size and distribution of brain-targeted liposomes and ζ-potential results in Examples and Comparative Examples

[0079] Figure 1 The particle size distribution diagram of the liposome preparations prepared in Example 1 and Comparative Example 1 is shown in Table 2 and Figure 1 It 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.

[0080] Example 7: Hemolytic evaluation Fresh whole blood from C57 mice was taken and placed in a sodium heparinized blood collection tube, then centrifuged at 1500 r / min for 5-10 min, the supernatant in the centrifuge tube was removed, PBS buffer was added to wash the red blood cells, and 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 by the method described in Example 1 was taken and gradually diluted with PBS buffer to make the concentration of the liposome preparation 25, 50, 100, 200 and 400 μg / mL. After taking 0.2 mL of the above liposome preparations of different concentrations and mixing with an equal volume of 2% red blood cell suspension, incubated at 37 ° C in a constant temperature shaker for 1 h, centrifuged at 10000 rpm for 10 min, and the supernatant was taken and the absorbance A was detected at a wavelength of 540 nm using an ELISA reader. The result of incubating 1% Triton (polyethylene glycol octylphenyl ether, TritonX-100) with erythrocytes was used as a positive control, that is, the hemolysis rate was 100%; the result of incubating PBS with erythrocytes was used as a negative control, that is, the hemolysis rate was 0%.

[0081] 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%.

[0082] The results are as follows Figure 2As shown, within the concentration range of 25-400 μg / mL of the liposome preparation prepared in Example 1, the liposome preparation did not cause obvious 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 has good blood compatibility.

[0083] Example 8: Anti-nonspecific protein adsorption assay The liposome preparations prepared by the methods described in Example 1 and Comparative Example 1 were incubated with bovine serum albumin (BSA) and histone (Histone) solutions in PBS (phosphate buffer) at pH = 7.4 at 37°C and 120 rpm, wherein the sample concentration was 0.1 mg / mL, and the concentrations of BSA and Histone were 0.1 mg / mL. After 2 hours, the mixed solution was taken out and centrifuged at 10,000 g for 15 min to precipitate the BSA and Histone adsorbed by the material. The concentrations of bovine serum albumin and histone in the supernatant were determined by a UV-visible near-infrared spectrophotometer, and their maximum absorbances at wavelengths of 280 nm and 275 nm were determined. Then, the adsorption amounts of BSA and Histone on the material sample were calculated based on the standard calibration curves of BSA and Histone. Each measurement was performed three times, and the results were averaged.

[0084] The results are as follows Figure 3 As shown, the liposome preparation prepared by the method described in Example 1 has an adsorption capacity of 17.25±7.06% for BSA and 20.05±9.05% for Histone. 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.

[0085] Example 9: Cytotoxicity Experiment The toxicity of brain-targeted liposome preparations to U-87 MG (human brain glioblastoma cells) and GL261 (mouse glioma cells) was investigated by MTT assay. U-87 MG and GL261 cells were cultured at 5×10 3 The cells were inoculated into 96-well plates at a concentration of 1000 cells / well and incubated at 37°C and 5% CO 2The cells were incubated in a cell culture incubator for 24 h. The liposome preparations (PCIGT@β-ELE, PCIT@β-ELE, PCGT@β-ELE, 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 obtain solutions with concentrations of 6.25, 12.5, 25, 50, 100, and 200 μg / mL of β-elemene. Similarly, the β-elemene (β-ELE) single administration group, temozolomide (TMZ) single administration group, second-generation dendritic glutamic acid modified temozolomide prodrug group (TMZ-G2E), second-generation dendritic glutamic acid modified temozolomide prodrug and β-elemene mixed administration group (β-ELE / TMZ-G2E Mix), in which β-ELE and TMZ-G2E were combined in a 3:1 molar ratio in the second-generation dendritic glutamic acid modified temozolomide prodrug and β-elemene mixed administration group, were diluted with DMEM complete medium to the same concentration (6.25, 12.5, 25, 50, 100 and 200 μg / mL), added to the cell culture plate and co-cultured for 48 hours. The culture medium was discarded, and 100 μL of PBS solution with a MTT concentration of 0.5 mg / mL was added to the 96-well plate in sequence and incubated in the cell culture incubator for another 4 hours. Discard the culture medium, add DMSO (100 μL) to the cell wells, shake evenly in an ELISA reader, and measure the absorbance OD 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%.

[0086] The results are as follows Figure 4 As shown, the IC values ​​of temozolomide alone group and 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 group treated with temozolomide and β-elemene alone, respectively; the IC 50 The values ​​were 108.04 μg / mL and 89.47 μg / mL, respectively. The IC values ​​of the brain-targeted liposomes prepared in Example 1 for GL261 cells were50 The value was 53.25 μg / mL, which was only 0.49 and 0.60 times of that of the group treated with temozolomide and β-elemene alone, respectively. The results showed that the brain-targeted liposomes prepared in Example 1 had significant inhibitory ability against both U-87 MG cells and GL261 cells. With the increase of the dosage, the inhibitory ability against tumor cells gradually increased, significantly reducing the IC 50 The values ​​indicated the coordinated antitumor effects of temozolomide and β-elemene.

[0087] Example 10: Cellular uptake experiment According to the preparation methods of the liposome preparations in Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4, elemene was replaced with doxorubicin (DOX) having red fluorescence to achieve intracellular tracing of the liposome preparations. U-87 MG cells and GL261 cells were cultured at 2×10 4 Each dish was inoculated in a glass-bottom dish and incubated at 37°C and 5% CO 2 The cells were cultured under the same conditions for 24 h, and the old culture medium was discarded. The above four DOX liposome preparations and DOX were diluted with DMEM complete culture medium and added to the glass-bottomed dish. The concentration of DOX was 10 μg / mL. The cells were incubated in a cell culture incubator for 2 or 4 h. Then the drug-containing culture medium was discarded, and Hoechst 33342 diluted in DMEM complete culture medium in advance was added. The final concentration of Hoechst 33342 was 10 μg / mL. The cells were incubated in an incubator for 20 min, and the old culture medium was discarded. The cells were washed three times with PBS and observed under a laser confocal microscope.

[0088] 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, because at 2 h, the liposome preparation had not been fully internalized by the cells. 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 liposome prepared by the method of comparative example 4 was negative, which repelled 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. PCGT@DOX liposome preparation can better enter tumor cells through glutathione transporter due to the presence of glutathione group; the strongest fluorescence was observed in the brain-targeted liposome preparation group (PCIGT@DOX) loaded with indole and glutathione modified with DOX, which is due to the synergistic effect of indole and glutathione, which enhances its transmembrane entry into cells. The above results show that the brain-targeted liposomes prepared by the method of Example 1 have excellent brain glioma targeting ability.

[0089] Example 11: Lysosomal escape experiment 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 Each dish was inoculated in a glass-bottom dish and incubated at 37°C and 5% CO 2 The cells were cultured under the same conditions for 24 h, the old culture medium was discarded, the above four DOX liposome preparations and DOX were diluted with DMEM complete culture medium and added to the dish, the concentration of DOX was 10 μg / mL, and they 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 culture 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 and observed under a laser confocal microscope.

[0090] The results are as follows Figure 6As shown in the figure, 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 was difficult to escape from the lysosome; the PCT@DOX group was difficult to enter the cell, so only weak green fluorescence was observed; extremely low green fluorescence was observed in the PCIT@DOX and PCIGT@DOX groups, because the indole residues inserted into the lysosomal lipid bilayer structure after entering the lysosome, destroying the lysosome; PCGT@DOX also observed a lower green fluorescence, which may be because the basic structure of liposomes is similar to that of biological membranes, so lysosomal escape can be performed by membrane fusion. The results show that the brain-targeted liposomes prepared by the method of Example 1 have excellent lysosomal escape performance.

[0091] Example 12: Brain endothelial cell transport performance experiment The ability of brain-targeted liposomes to cross the blood-brain barrier and enter the brain tissue was evaluated by the endocytosis experiment of mouse brain endothelial cells (bEnd.3). 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 liposome preparation. bEnd.3 cells were cultured at 2×10 4 Each dish was inoculated in a glass-bottom dish and incubated at 37°C and 5% CO 2 The cells were cultured under the same conditions for 24 h, the old culture medium was discarded, the above four DOX liposome preparations and pure DOX were diluted with DMEM complete culture medium and added to the glass-bottomed dish, the concentration of DOX was 10 μg / mL, and they 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 culture 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 and observed under a laser confocal microscope.

[0092] The results are as follows Figure 7As shown, in bEnd.3 cells, for each control group, the red fluorescence intensity at 2 h was greater than 4 h, because at 2 h, the liposome preparation had not been fully internalized by the endothelial cells. Because the indole residues and glutathione groups can target the amino acid receptors and glutathione transporters highly expressed in brain endothelial cells, respectively, stronger fluorescence was observed in both the PCIT@DOX and PCGT@DOX groups than in the DOX group alone, proving that the liposome preparations modified with indole residues and glutathione groups can help small molecules to more efficiently pass through the blood-brain barrier through the transport of endothelial cells. Among them, the strongest red fluorescence was observed in the PCIGT@DOX group, because the presence of the dual targeting groups further enhanced the endothelial cells' endocytosis of brain-targeted liposomes. The results show that the brain-targeted liposome preparation prepared in Example 1 has excellent blood-brain barrier penetration ability.

[0093] Example 13: Establishing an in vitro blood-brain barrier model to evaluate the ability of liposomes to penetrate the blood-brain barrier According to the preparation method of the 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 a 24-well Transwell for monolayer culture. 5 GL261 cells were inoculated in 800 μL of culture medium, which was injected into the lower chamber of the Transwell. When the monolayer anti-endothelial electrical resistance (TEER) reached 200 Ω·cm² or more, the blood-brain barrier model could be used for the next experiment. The above four DOX liposome preparations and DOX alone were diluted with DMEM complete medium and added to the dish to make the concentration of DOX 20 μg / mL. They were incubated in a cell culture incubator for 4 h, the drug-containing medium was discarded, and Hoechst 33342 diluted in DMEM complete medium was added to a final concentration of 10 μg / mL. The old medium was discarded, and the dish was washed 3 times with PBS and observed under an inverted fluorescence microscope.

[0094] The results are as follows Figure 8 As 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 number of indole residues when penetrating the bEnd.3 tight junction. The PCGT@DOX group and the PCIGT@DOX group observed stronger red fluorescence under the same glutathione molar equivalent, which further proved that indole residues can destroy membrane structure and enhance lysosomal escape. The results showed that the brain-targeted liposome preparation prepared in Example 1 above can cross the in vitro blood-brain barrier model and enter glioma cells.

[0095] Example 14: In vivo biodistribution experiment 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) for easy in vivo tracing to prepare Dir-labeled brain-targeted liposomes, and the 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 (1, 2, 3, 4, 6, 8, 10, 24, and 48 hours) after injection. Finally, the heart, lung, liver, kidney, spleen, and brain were collected, and ex vivo imaging was performed 48 hours after injection.

[0096] The results are as follows Fig. 9 As shown in the figure, the brain glioma mice injected with PCIGT@Dir showed stronger fluorescence in the brain than the PCT@Dir group. In addition, a certain enrichment was observed in the mouse skull of the PCIGT@Dir group 1 hour after injection, and reached the maximum value at 24 hours after injection. The sustained effect lasted for 48 hours, and the in vitro distribution results were consistent with the in vivo distribution. The results show that the brain-targeted liposome preparation prepared in Example 1 can effectively cross the blood-brain barrier and continuously accumulate in brain tissue.

[0097] Example 15: In vivo anti-glioma experiment A C57BL / 6 mouse model bearing brain glioma was constructed, and the indole and glutathione modified brain-targeted liposome formulation loaded with elemene and temozolomide prodrug prepared in Example 1 was evaluated for its anti-glioma performance in vivo. Eighteen tumor-bearing C57BL / 6 mice were randomly divided into three groups, with six mice in each group. They were (1) saline control group (referred to as 0.9% NaCl group); (2) β-elemene injection group (referred to as β-ELE group); and (3) PCIGT@β-ELE group. Drugs were 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, tumor-bearing mice were subjected to bioluminescence imaging on days 0, 5, 10, and 15 to evaluate tumor inhibition.

[0098] The results are as follows Fig.10 As shown in the figure, after 15 days of treatment, the tumor fluorescence intensity of the PCIGT@β-ELE group was the smallest compared with the other groups. The β-ELE group also had a certain inhibitory effect on brain glioma, but the inhibitory effect was relatively weak. The results showed that the indole and glutathione-modified brain-targeted liposome preparation loaded with elemene and glutamic acid-modified temozolomide prodrug had a good anti-glioma effect.

[0099] It can be seen from the above examples that 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 synergy 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 liposome preparation 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.

[0100] Although the above embodiment describes the present invention in detail, 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 protection scope of the present invention.

Claims

1. A liposome, characterized in that: The liposome membrane is modified with glutathione groups and indole residues.

2. The liposome according to claim 1, characterized in that The liposome comprises the following raw materials in parts by weight: 2-8 parts of indole-oleylamine, 20-40 parts of DSPE-PEG-glutathione, 80-120 parts of phospholipids and 2-6 parts of cholesterol; The indole-oleylamine is prepared by condensing oleylamine and indole-2-carboxylic acid.

3. The liposome according to claim 2, characterized in that The phospholipids include one or more of soybean lecithin, egg yolk lecithin, hydrogenated lecithin and synthetic lecithin; The raw material for preparing the DSPE-PEG-glutathione includes polyethylene glycol, and the weight average molecular weight of the polyethylene glycol is 1000-10000 Da.

4. Use of the liposome according to any one of claims 1 to 3 in the preparation of a brain-targeted liposome preparation.

5. A liposome preparation, characterized in that: The liposome comprising any one of claims 1 to 3; Also included are drug molecules encapsulated in the vesicles of the liposomes, wherein the drug molecules include glutamic acid-modified temozolomide and elemene.

6. The liposome preparation according to claim 5, characterized in that The glutamic acid-modified temozolomide comprises one or more of the following chemical structures: 。 7. The liposome preparation according to claim 5, characterized in that The elemene includes one or more of α-elemene, β-elemene, γ-elemene and δ-elemene.

8. The liposome preparation according to any one of claims 5 to 7, characterized in that The liposome preparation comprises the following raw materials in parts by weight: 2-8 parts of indole-oleylamine, 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.

9. The method for preparing the liposome preparation according to any one of claims 5 to 8, 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 hydrating medium to obtain the liposome preparation.

10. Use of the liposome preparation according to any one of claims 5 to 8 or the liposome preparation prepared by the preparation method according to claim 9 in the preparation of brain-targeted anticancer drugs.

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