Brain glioma treatment effect evaluation method and kit based on dual-mediated liposome drug delivery system

Abstract

The invention provides a brain glioma treatment effect evaluation method based on a dual-mediated liposome drug delivery system and a kit. The evaluation method comprises the following steps: step 1, obtaining the dual-mediated liposome drug delivery system; step 2, respectively evaluating the transport mechanisms of the dual-mediated liposome drug delivery system in BMVEC cells and C6 cells; 3, obtaining a BBB / C6 co-culture model, and evaluating the transport condition of the dual-mediated liposome drug delivery system in the BBB / C6 co-culture model; and 4, according to the results of the step 2 and the step 3, obtaining an evaluation result of the double-mediated liposome drug delivery system on the treatment effect of the brain glioma. According to the invention, a method capable of accurately and efficiently evaluating the treatment effect of brain glioma is constructed by researching the transport pathway of the dual-mediated brain-targeted liposome, a cell internalization mechanism, a cross-BBBs transport pathway, in-vivo targeting and neurotoxicity.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method and a kit for evaluating the therapeutic effect of glioma based on a dual-mediated liposome drug delivery system. Background Art

[0002] The blood-brain barrier (BBB) ​​is a physiological barrier that restricts most drugs from entering the brain. The design of a rational and efficient brain-targeted drug delivery system will hopefully achieve brain delivery of existing drugs. In the past 20 years, liposomes have made great progress as brain-targeted drug delivery systems in drug brain delivery. Based on the structural and functional characteristics of the BBB, current research is mainly focused on receptor-mediated active transport. The high specificity and selectivity of receptors promote the concentration of drugs in target organs. However, due to the saturation of receptors expressed by cells and the difficulty of drugs escaping from lysosomes after receptor-mediated introduction into cells, the drugs are limited from reaching the real target to exert their efficacy. In fact, in addition to expressing abundant receptors, the BBB also exhibits abundant negative charges. Therefore, adsorption mediation using positively charged drug delivery systems and negatively charged BBB is an effective way to cross the BBB. At the end of the 20th century, cell membrane penetrating peptides (CPPs) were discovered to be rich in arginine residues, positively charged under normal physiological and lysosomal acidic conditions, and have significant cell penetration and lysosomal escape capabilities. However, CPPs lack tissue and cell selectivity, which brings about thorny problems such as low target cell uptake efficiency, poor absorption / distribution in vivo, low bioavailability and toxicity, which seriously limits their application as drug carriers. In view of the advantages and limitations of receptor-mediated and adsorption-mediated, combining the two to complement each other is an effective strategy to achieve brain-targeted drug delivery. Studies have shown that brain-targeted liposomes can increase the blood-brain barrier permeability of drugs through receptor-mediated active transport. However, liposomes mediated by a single receptor are difficult to escape from lysosomes, which limits their application in brain drug delivery. In addition to expressing abundant receptors, the BBB also exhibits abundant negative charges. Positively charged cell penetrating peptides (CPPs) can play a cell membrane penetration role through electrostatic interactions and have lysosomal escape capabilities. However, CPP lacks tissue and cell selectivity, and has serious toxicity and side effects when used alone, which limits its application in the construction of brain-targeted liposomes. Dual-mediated brain-targeted liposomes designed by combining receptor mediation and adsorption mediation are expected to break through the limitations of single receptor mediation or adsorption mediation, significantly enhance the BBB permeability of brain-targeted liposomes, and have important research value. The construction of dual-mediated liposomes mainly includes two methods: one is to modify the target ligand and CPP on the surface of liposomes separately to form dual-ligand modified liposomes, and the other is to first couple the target ligand to CPP, and then modify the coupling polypeptide on the surface of liposomes to form single-ligand liposomes.

[0003] Kumar introduced positively charged R9 at the C-terminus of rabies virus glycoprotein 29 (RVG29), carried siRNA into the brain and caused effective gene silencing (Kumar P, Wu H, McBride JL, Jung KE, Kim MH, Davidson BL, Lee SK, Shankar P, Manjunath N. Transvascular delivery of small interfering RNA to the central nervous system [J]. Nature, 2007, 448 (149): 39-43). However, there is currently a lack of systematic research on the feasibility of direct coupling of targeting peptides and CPP. Will the direct coupling of CPP with targeting peptides reduce its targeting ability? Is the transport mechanism of this single ligand-modified liposome on the blood-brain barrier different from the transport mechanism of dual-ligand liposomes? Is safety affected? These are all problems to be solved. The present invention is a study based on the aforementioned problems. Summary of the invention

[0004] The purpose of the present invention is to provide a method and a kit for evaluating the therapeutic effect of glioma based on a dual-mediated liposome drug delivery system, by comparing the previously constructed single-ligand liposomes (RVGPR9-SSL, i.e., PTB-SSL, disclosed in CN113880913A) and the dual-ligand liposomes (RVGP-R9-SSL, disclosed in CN113880913A), including: the uptake mode, transport mechanism, in vivo distribution targeting and safety evaluation of the two drug delivery systems on BMVEC cells and C6 cells, thereby providing important data support for the construction and application of dual-mediated brain-targeted liposomes.

[0005] In order to achieve the above technical objectives, the technical solutions adopted in this application are as follows:

[0006] In a first aspect, the present invention provides a method for evaluating the therapeutic effect of glioma based on a dual-mediated liposome drug delivery system, comprising the following steps:

[0007] Step 1, obtaining a dual-mediated liposome drug delivery system;

[0008] Step 2, respectively evaluating the transport mechanism of the dual-mediated liposome drug delivery system in BMVEC cells and C6 cells;

[0009] Step 3, obtaining a BBB / C6 co-culture model, and evaluating the transport of the dual-mediated liposome drug delivery system in the BBB / C6 co-culture model;

[0010] Step 4: Based on the results of step 2 and step 3, the evaluation results of the dual-mediated liposome drug delivery system for the treatment of brain glioma are obtained.

[0011] Preferably, in step 1, the dual-mediated liposome drug delivery system is RVGPR9-SSL and RVGP-R9-SSL.

[0012] Preferably, in step 2, chlorpromazine is used as a clathrin inhibitor, filipin is used as a caveolae inhibitor, RVG29 is used as an AChR inhibitor, and sodium azide is used as an energy inhibitor to evaluate the transport mechanism of the dual-mediated liposome drug delivery system in BMVEC cells and C6 cells, respectively.

[0013] Preferably, step 2 comprises the following steps:

[0014] Step 2-1, using DMEM medium without phenol red to prepare inhibitor reserve solutions of chlorpromazine, philipin, sodium azide, and RVG29 respectively; and using DMEM medium without phenol red to prepare coumarin-encapsulated SSL, RVGPR9-SSL, and RVGP-R9-SSL liposome test solutions respectively; the four inhibitor reserve solutions and the three test solutions are mixed respectively to form 12 groups of inhibitor-containing liposome solutions;

[0015] Step 2-2, incubating BMVEC cells or C6 cells with chlorpromazine, philipid, sodium azide and RVG29 respectively, and then incubating the BMVEC cells or C6 cells after the incubation with a liposome solution containing an inhibitor, the culture solution after the incubation is used as the test group, and the test solution without the inhibitor is used as the control group, and the uptake of each group of experimental solutions by BMVEC cells or C6 cells is detected by flow cytometry;

[0016] Step 2-3, using the fluorescent probe lyso-tracker Red to label the lysosomes of BMVEC cells, and using the fluorescent probe ER-tracker-Red to label the endoplasmic reticulum of BMVEC cells; then incubating the coumarin-loaded SSL, R9-SSL, RVGP-SSL, RVGPR9-SSL and RVGP-R9-SSL with the BMVEC cells labeled with the fluorescent probes, and observing the colocalization of each liposome solution with the lysosomes and endoplasmic reticulum of the BMVEC cells by laser confocal microscopy;

[0017] Step 2-4, using the fluorescent probe lyso-tracker Red to label the lysosomes of C6 cells, and using the fluorescent probe ER-tracker-Red to label the endoplasmic reticulum of C6 cells; then incubating SSL, RVGPR9-SSL and RVGP-R9-SSL loaded with doxorubicin with the C6 cells labeled with the fluorescent probes, and observing the co-localization of each liposome solution with the lysosomes and endoplasmic reticulum of C6 cells by laser confocal microscopy;

[0018] Step 2-5, measuring the Zeta potential of the liposome solution containing the inhibitor at pH 7.4 and pH 6.5.

[0019] Preferably, step 3 comprises the following steps:

[0020] Step 3-1, obtaining an in vitro BBB model;

[0021] Step 3-2, co-culturing C6 cells with the in vitro BBB model to obtain a BBB / C6 co-culture model;

[0022] Step 3-3, adding the dual-mediated liposome drug delivery system to the BBB / C6 co-culture model, and then evaluating the uptake results of the dual-mediated liposome drug delivery system in the BBB / C6 co-culture model.

[0023] A kit for evaluating the therapeutic effect of glioma based on a dual-mediated liposome drug delivery system, characterized in that it comprises:

[0024] BMVEC cell evaluation unit;

[0025] C6 Cell Evaluation Unit;

[0026] BBB / C6 co-culture model evaluation unit.

[0027] Preferably, the BMVEC cell evaluation unit comprises: BMVEC cells, a reserve inhibitor solution, a fluorescent dye for marking the dual-mediated liposome drug delivery system, and a fluorescent probe for marking BMVEC cells.

[0028] Preferably, the C6 cell evaluation unit comprises: C6 cells, a reserve inhibitor solution, a fluorescent dye for marking a dual-mediated liposome drug delivery system, a fluorescent probe for marking C6 cells, and an anti-tumor drug; preferably, the anti-tumor drug is doxorubicin.

[0029] Preferably, the BBB / C6 co-culture model evaluation unit comprises: a BBB / C6 co-culture model.

[0030] Preferably, the preparation method of the inhibitor reserve solution is: the inhibitors chlorpromazine, philipin, sodium azide, and RVG29 are respectively prepared into the inhibitor reserve solution using a DMEM culture medium without phenol red.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention constructs a method that can accurately and efficiently evaluate the therapeutic effect of brain glioma by studying the transport pathways, cellular internalization mechanisms, and trans-BBBs transport pathways, in vivo targeting and neurotoxicity of two dual-mediated brain-targeted liposomes RVGPR9-SSL and RVGP-R9-SSL constructed in the early stage. The results show that different modification patterns will affect the cellular internalization mechanism and transport pathway of dual-mediated brain-targeted liposomes, thereby affecting their BBB permeability and brain targeting efficiency. RVGPR9-SSL can be effectively transported across the BBB, has good brain targeting ability, can bypass lysosomal transport and target tumor cell nuclei. Therefore, RVGPR9-SSL is an ideal brain-targeted drug delivery system, which is expected to play a good therapeutic role in glioma. In addition, the present invention provides important method guidance and data support for the construction of subsequent dual-mediated brain-targeted liposomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The results of the study on the transport mechanism of liposomes in BMVEC cells in Example 1 of the present invention are shown in Figures a and c, which show the co-localization results of five liposomes with lysosomes in BMVEC cells; Figure b shows the effect of inhibitors on the cell uptake of SSL, RVGPR9-SSL and RVGP-R9-SSL in BMVEC cells; and Figure d shows the quantitative results of the co-localization of five liposomes with lysosomes in BMVEC cells. (Scale bar: 10 μm; n=3, p value: *p<0.05; **p<0.0001).

[0034] Figure 2 The results of the study on the transport mechanism of liposomes in BMVEC cells in Example 1 of the present invention are shown in Figures a and b, respectively, for the co-localization results of the endoplasmic reticulum of BMVEC cells; Figure c is the quantitative results of the co-localization of five liposomes with the endoplasmic reticulum in BMVEC cells. (Scale bar: 10 μm; n=3, p value: *p<0.05; **p<0.01; ***p<0.0001).

[0035] Figure 3 Schematic diagram of the transport mechanism of RVGPR9-SSL and RVGP-R9-SSL in BMVEC cells in Example 1 of the present invention.

[0036] Figure 4The results of the study on the transport mechanism of liposomes in C6 cells in Example 2 of the present invention are shown in Figure a, wherein Figure a shows the effect of inhibitors on the cell uptake of SSL (Cou-6), RVGPR9-SSL (Cou-6) and RVGP-R9-SSL (Cou-6); Figure b and Figure c show the co-localization results of RVGPR9-SSL (Cou-6) and RVGP-R9-SSL (Cou-6) with C6 cell lysosomes; Figure d and Figure e show the co-localization results of RVGPR9-SSL (Cou-6) and RVGP-R9-SSL (Cou-6) with the endoplasmic reticulum of C6 cells. (Scale bar: 10 μm; n=3, p value: *p<0.001; ****p<0.0001).

[0037] Figure 5 The figure is a result diagram of the predicted therapeutic effect of the liposomes encapsulating the chemotherapy drug DOX in Example 2 of the present invention, wherein Figure a and Figure b are the co-localization results of SSL (DOX), RVGPR9-SSL (DOX) and RVGP-R9-SSL (DOX) with the nucleus of C6 cells. (Scale bar: 10 μm; n=3, p value: *p<0.05; **p<0.0001).

[0038] Figure 6 The uptake of the liposomes in the BBB / C6 co-culture model in Example 3 of the present invention, wherein, Figure a is a schematic diagram of the BBB / C6 co-culture model; Figure b is the tight junctions between BMVEC cells observed by SEM (×1500); Figure c is the uptake of three types of Cou-6 liposomes in the BBB / C6 co-culture model; Figure d is the average of the fluorescence intensity of the uptake of the three types of liposomes in the BBB / C6 co-culture model. (n=3, p value: *p<0.05; **p<0.01; ***p<0.0001). DETAILED DESCRIPTION

[0039] In the embodiments of the present invention, the preparation methods of SSL and SSL (Cou-6) are as follows:

[0040] Preparation of SSL: According to the molar ratio of 100:50:8, accurately weigh appropriate amount of soybean lecithin (SPC), cholesterol (Chol), and DSPE-PEG2000 and add them to an eggplant-shaped bottle, then add appropriate amount of chloroform to dissolve, place on a vortex oscillator to shake and mix evenly, place on a rotary evaporator, spin dry the organic solvent in a 37°C constant temperature water bath until a uniform lipid film is formed at the bottom of the eggplant-shaped bottle, and continue to spin dry for 30 minutes to remove the residual organic solvent. Then add an appropriate amount of PBS buffer (pH = 7.4, 0.1 mol / L) to the eggplant-shaped bottle, place on a vortex oscillator for vortexing, and then place in a 37°C water bath for ultrasonication for 30 minutes until complete hydration, and SSL is obtained;

[0041] Preparation of SSL (Cou-6): Accurately weigh a certain amount of SPC, Chol, DSPE-PEG2000, and an appropriate amount of coumarin-6 (Cou-6), add them to an eggplant-shaped bottle in a certain ratio (SPC: Chol: DSPE-PEG2000 = 100: 50: 8 mol / mol; Coumarin-6: SPC = 1.5: 2000, w / w), then add an appropriate amount of chloroform to dissolve, place on a vortex oscillator to oscillate and mix evenly, place on a rotary evaporator, spin dry the organic solvent in a 37°C constant temperature water bath until a uniform lipid film is formed at the bottom of the eggplant-shaped bottle, and continue to spin dry for 30 minutes to remove the residual organic solvent. Then add an appropriate amount of PBS buffer (pH = 7.4, 0.1 mol / L) to the eggplant-shaped bottle, place on a vortex oscillator for vortex oscillation, and then place in a 37°C water bath for ultrasonic 30 minutes until hydration is complete to obtain SSL (Cou-6).

[0042] In the embodiments of the present invention, the preparation methods of RVGPR9-SSL (Cou-6) and RVGP-R9-SSL (Cou-6) refer to paragraphs

[0091] -

[0093] of patent CN113880913A.

[0043] In an embodiment of the present invention, the preparation methods of R9-SSL, R9-SSL(Cou-6) and RVGP-SSL, RVGP-SSL(Cou-6) are as follows:

[0044] (1) Preparation of R9-SSL: Precisely weigh a certain amount of SPC, Chol, DSPE-PEG1000, and DSPE-PEG1000-R9, and add them to an eggplant-shaped bottle in a certain ratio (SPC: Chol: DSPE-PEG1000: DSPE-PEG1000-R9 = 100: 50: 4: 4 mol / mol), then add an appropriate amount of chloroform to dissolve, place on a vortex oscillator to oscillate and mix evenly, place on a rotary evaporator, spin dry the organic solvent in a 37°C constant temperature water bath until a uniform lipid film is formed at the bottom of the eggplant-shaped bottle, and continue to spin dry for 30 minutes to remove the residual organic solvent. Then add an appropriate amount of PBS buffer (pH = 7.4, 0.1 mol / L) to the eggplant-shaped bottle, place on a vortex oscillator for vortexing, and then place in a 37°C water bath for ultrasonication for 30 minutes until complete hydration, thereby obtaining R9-SSL.

[0045] (2) Preparation of R9-SSL (Cou-6): Accurately weigh a certain amount of SPC, Chol, DSPE-PEG1000, DSPE-PEG1000-R9 and an appropriate amount of coumarin-6 (Cou-6), and add them into an eggplant-shaped bottle in a certain ratio (SPC: Chol: DSPE-PEG1000: DSPE-PEG1000-R9 = 100: 50: 4: 4 mol / mol; Coumarin-6: SPC = 1.5: 2000, w / w). Then, add an appropriate amount of chloroform to dissolve, place on a vortex oscillator to oscillate and mix evenly, place on a rotary evaporator, and spin dry the organic solvent in a constant temperature water bath at 37°C until a uniform lipid film is formed at the bottom of the eggplant-shaped bottle. Continue to spin dry for 30 min to remove the residual organic solvent. Then, an appropriate amount of PBS buffer (pH=7.4, 0.1 mol / L) was added to the eggplant-shaped bottle, vortexed on a vortex oscillator, and then ultrasonicated in a 37° C. water bath for 30 min until complete hydration, to obtain R9-SSL (Cou-6).

[0046] (3) Preparation of RVGP-SSL: Precisely weigh a certain amount of SPC, Chol, DSPE-PEG2000, and DSPE-PEG2000-RVGP, and add them to an eggplant-shaped bottle according to SPC: Chol: DSPE-PEG2000: DSPE-PEG2000-RVGP = 100: 50: 6: 2, mol / mol). Then add an appropriate amount of chloroform to dissolve, place on a vortex oscillator to oscillate and mix evenly, place on a rotary evaporator, and spin dry the organic solvent in a constant temperature water bath at 37°C until a uniform lipid film is formed at the bottom of the eggplant-shaped bottle. Continue to spin dry for 30 minutes to remove the residual organic solvent. Then add an appropriate amount of PBS buffer (pH = 7.4, 0.1 mol / L) to the eggplant-shaped bottle, place on a vortex oscillator for vortexing, and then place in a 37°C water bath for ultrasonication for 30 minutes until complete hydration, thereby obtaining RVGP-SSL.

[0047] (4) Preparation of RVGP-SSL (Cou-6): Accurately weigh a certain amount of SPC, Chol, DSPE-PEG2000, DSPE-PEG2000-RVGP and an appropriate amount of coumarin-6 (Cou-6), and add them into an eggplant-shaped bottle according to SPC: Chol: DSPE-PEG2000: DSPE-PEG2000-RVGP = 100: 50: 6: 2 mol / mol; Coumarin-6: SPC = 1.5: 2000, w / w). Then add an appropriate amount of chloroform to dissolve, place on a vortex oscillator to oscillate and mix evenly, place on a rotary evaporator, and spin dry the organic solvent in a constant temperature water bath at 37°C until a uniform lipid film is formed at the bottom of the eggplant-shaped bottle. Continue spinning for 30 minutes to remove the residual organic solvent. Then, an appropriate amount of PBS buffer (pH=7.4, 0.1 mol / L) was added to the eggplant-shaped bottle, vortexed on a vortex oscillator, and then ultrasonicated in a 37°C water bath for 30 min until complete hydration, to obtain RVGP-SSL (Cou-6).

[0048] The preparation method of DSPE-PEG1000-R9 is as follows: R9 peptide and PEGylated active phospholipid (DSPE-PEG1000-NHS) are dissolved in anhydrous dimethylformamide (DMF) respectively, and 200 μL of triethylamine reagent is added to the above R9 peptide solution to adjust the pH of the reaction solution to 7.4. Then the DSPE-PEG1000-NHS solution is slowly and evenly added to the R9 solution (DSPE-PEG1000-NHS: R9 = 1:2, mol / mol), and then an appropriate amount of catalyst DMAP is added, and the reaction mixture is placed on a magnetic stirrer and reacted at room temperature for 36 hours under light shielding. After the reaction is completed, the reaction mixture is taken into a dialysis bag (molecular weight cutoff is 3500Da), placed in 1000mL deionized water for 36 hours, and the dialysis external solution is changed every 6 hours. In order to remove the reactants, triethylamine and solvent that have not reacted completely. After the dialysis is completed, the dialyzate is pre-frozen in a -20°C refrigerator for 12 hours, and then the pre-frozen sample is placed at -40°C and 0.22 mbar for freeze-drying for 48 hours to obtain a white loose solid sample.

[0049] The preparation method of DSPE-PEG2000-RVGP is as follows: RVGP peptide and DSPE-PEG2000-NHS are dissolved in anhydrous dimethyl sulfoxide (DMSO) respectively. 200 μL of triethylamine reagent is added to the RVGP polypeptide solution to adjust the pH of the reaction solution to 7.4, and then the DSPE-PEG2000-NHS solution is slowly and evenly added to the RVGP polypeptide solution (DSPE-PEG2000-NHS:RVGP=1:2, mol / mol), and then an appropriate amount of catalyst DMAP is added, and the reaction mixture is placed on a magnetic stirrer and reacted at room temperature for 36 hours under light shielding. After the reaction is completed, the reaction mixture is placed in a dialysis bag (molecular weight cutoff is 3500Da), placed in 1000mL deionized water for 36 hours, and the dialysis external liquid is changed every 6 hours. In order to remove the reactants, triethylamine and solvent that have not reacted completely. After the dialysis is completed, the dialyzate is pre-frozen in a -20°C refrigerator for 12 hours, and then the pre-frozen sample is placed at -40°C and 0.22 mbar for freeze-drying for 48 hours to obtain a white loose solid sample.

[0050] In an embodiment of the present invention, the preparation method of liposomes (SSL (DOX), RVGPR9-SSL (DOX), RVGP-R9-SSL (DOX)) loaded with the chemotherapy drug doxorubicin (DOX) comprises:

[0051] (1) Preparation of SSL (DOX): Accurately weigh appropriate amounts of SPC, Chol, and DSPE-PEG2000, add them to an eggplant-shaped bottle in a certain ratio (SPC: Chol: DSPE-PEG2000 = 100:50:8, mol / mol), dissolve them with chloroform, vortex mix them, place them on a rotary evaporator, and evaporate the organic solvent in a 37°C water bath until a uniform lipid film is formed at the bottom of the eggplant-shaped bottle, and then continue to vacuum for 30 minutes to remove the remaining organic solvent. Then add 2 mL of pH = 4.0 citric acid buffer (300 mM) to the eggplant-shaped bottle, place it on a vortex oscillator and vortex it, and then place it in a 37°C water bath for 30 minutes for ultrasonication until it is completely hydrated, thereby obtaining blank SSL liposomes. Subsequently, the pH value of the blank SSL liposomes was adjusted to 7.5-7.8 with 0.5 M sodium carbonate solution. Under preheating in a 50° C. water bath, a DOX aqueous solution (DOX / SPC=1:20, w / w) was added and stirred for 30 min to allow the liposomes to fully encapsulate doxorubicin, thereby obtaining SSL (DOX).

[0052] (2) Preparation of RVGPR9-SSL (DOX): Accurately weigh appropriate amounts of SPC, Chol, DSPE-PEG2000, and DSPE-PEG2000-RVGPR9, and add them into an eggplant-shaped bottle in a certain ratio (SPC: Chol: DSPE-PEG2000: DSPE-PEG2000-RVGPR9 = 100:50:6:2, mol / mol). For other specific preparation methods, please refer to the above-mentioned preparation method of SSL (DOX).

[0053] (3) Preparation of RVGP-R9-SSL (DOX): Accurately weigh appropriate amounts of SPC, Chol, DSPE-PEG2000, DSPE-PEG2000-RVGP, and DSPE-PEG1000-R9, and add them into an eggplant-shaped bottle in a certain proportion (SPC: Chol: DSPE-PEG2000: DSPE-PEG2000-RVGP: DSPE-PEG1000-R9 = 100:50:2:2:4, mol / mol). For other specific preparation methods, please refer to the above-mentioned preparation method of SSL (DOX).

[0054] Preparation of DSPE-PEG2000-RVGPR9: RVGPR9 peptide and DSPE-PEG2000-NHS were dissolved in anhydrous dimethyl sulfoxide (DMSO) respectively. 200 μL of triethylamine reagent was added to the RVGPR9 peptide solution to adjust the pH of the reaction solution to 7.4, and then the DSPE-PEG2000-NHS solution was slowly and evenly added to the RVGPR9 peptide solution (DSPE-PEG2000-NHS:RVGPR9=1:2, mol / mol), and then an appropriate amount of catalyst DMAP was added. The reaction mixture was placed on a magnetic stirrer and reacted at room temperature for 36 hours under light shielding. After the reaction was completed, the reaction mixture was placed in a dialysis bag (molecular weight cutoff of 3500Da) and dialyzed in 1000mL deionized water for 36 hours, and the dialysis external solution was changed every 6 hours. In order to remove the reactants, triethylamine and solvent that were not completely reacted. After the dialysis is completed, the dialyzate is pre-frozen in a -20°C refrigerator for 12 hours, and then the pre-frozen sample is placed at -40°C and 0.22 mbar for freeze-drying for 48 hours to obtain a white loose solid sample.

[0055] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, which are intended to explain the present invention rather than to limit it.

[0056] Example 1

[0057] This example uses an uptake inhibition test and an organelle co-localization experiment to investigate the transport mechanism of RVGPR9-SSL and RVGP-R9-SSL in BMVEC cells, the main component of the BBB. We selected chlorpromazine as a clathrin inhibitor, philipin as a caveolae inhibitor, RVG29 as an AChR inhibitor, and sodium azide as an energy inhibitor to study the inhibitory effects of the four inhibitors on the uptake of RVGPR9-SSL and RVGP-R9-SSL in BMVEC cells. The details are as follows:

[0058] The inhibitors chlorpromazine, philipin, sodium azide, and RVG29 were prepared into four inhibitor stock solutions with final concentrations of 30 μmol / L, 10 μmol / L, 0.5 mmol / L, and 0.1 mg / mL, respectively, using DMEM medium without phenol red. Coumarin-encapsulated liposome SSL (Cou-6), RVGPR9-SSL (Cou-6), and RVGP-R9-SSL (Cou-6) (the SPC concentration of the three liposome solutions was 2 mg / ml) were diluted with DMEM medium without phenol red to a test solution without inhibitors with a final SPC concentration of 0.2 mg / mL and a final Cou-6 concentration of 150 ng / mL. Coumarin-loaded liposome SSL (Cou-6), RVGPR9-SSL (Cou-6) and RVGP-R9-SSL (Cou-6) (the SPC concentration of the three liposome solutions was 2 mg / ml) were diluted with different inhibitor stock solutions to obtain inhibitor-containing test solutions with a final SPC concentration of 0.2 mg / mL and a final Cou-6 concentration of 150 ng / mL. Both the inhibitor stock solution and the test solution were sterilized by filtration with a 0.22 μm filter membrane.

[0059] BMVEC cells (purchased outsourced) were incubated with inhibitors (chlorpromazine, philipin, sodium azide and RVG29) for 30 minutes; the BMVEC cells after incubation were incubated with SSL (Cou-6), RVGPR9-SSL (Cou-6) or RVGP-R9-SSL (Cou-6) solutions containing inhibitors for 1 hour. The test solution without inhibitors was used as the control group. Flow cytometry was used to detect the uptake of each group of solutions by BMVEC cells. Next, the lysosomes of BMVEC cells were labeled with the fluorescent probe lyso-tracker Red, and the endoplasmic reticulum of BMVEC cells was labeled with the fluorescent probe ER-tracker-Red. Then SSL (Cou-6), R9-SSL (Cou-6), RVGP-SSL (Cou-6), RVGPR9-SSL (Cou-6) and RVGP-R9-SSL (Cou-6) were incubated with fluorescently labeled BMVEC cells. After 1, 2, and 4 hours, laser confocal microscopy was used to observe the co-localization of each liposome solution with the lysosome and endoplasmic reticulum of BMVEC cells.

[0060] The experimental results are as follows Figure 1 and Figure 2 As shown, Figure 1 Figure b shows that SSL (Cou-6) is mainly taken up into BMVEC cells through clathrin-mediated uptake, RVGPR9-SSL (Cou-6) is mainly taken up into cells through receptor-mediated and caveolin-mediated uptake, RVGP-R9-SSL (Cou-6) is mainly taken up into cells through receptor-mediated and clathrin-mediated uptake, and the uptake of the three liposomes is energy-dependent.

[0061] The results showed that RVGPR9-SSL liposomes colocalized with lysosomes or endoplasmic reticulum of BMVEC cells. Figure 1 Figures a and c, and Figure 2 In Figures a and b), the co-localization rate of RVGPR9-SSL with the endoplasmic reticulum was significantly lower than that with the lysosome, indicating that RVGPR9-SSL was mainly transported through the endoplasmic reticulum pathway, while RVGP-R9-SSL was mainly transported through the lysosome pathway ( Figure 1 Figures a, c, and d, and Figure 2 In addition, compared with SSL, the RVGP-SSL modified with ligand RVGP showed significantly increased aggregation in lysosomes, but no lysosomal escape within 4 hours ( Figure 1 Compared with SSL and RVGP-SSL, liposomes modified with cell-penetrating peptide R9 (R9-SSL, RVGPR9-SSL, and RVGP-R9-SSL) showed a certain ability to escape lysosomes ( Figure 1 Figures a, c and d in the middle).

[0062] The above results indicate that changes in the way the ligand is modified on the liposome surface will change the uptake mechanism of the liposome. RVGPR9-SSL mainly enters BMVEC cells through receptor-mediated and caveolin-mediated modes, which may be related to the overall effect of RVGPR9. On the other hand, RVGP-R9-SSL is mainly taken up into cells through receptor-mediated and clathrin-mediated modes, which may be related to the fact that RVGP and R9 are connected to two PEG chains respectively. When approaching BMVEC cells, the RVGP connected to the long-chain PEG first binds to the nAChR, triggering the receptor-mediated endocytosis process. When RVGP interacts with the receptor, the distance between the R9 connected to the short-chain PEG and the BMVEC cell is shortened, thereby triggering the endocytosis pathway related to the action of R9, that is, the clathrin-mediated uptake mode. This study shows that the uptake mechanism of liposomal nanoparticles affects their intracellular transport mechanism. Nanoparticles enter cells through clathrin- or caveolin-mediated uptake mechanisms, and their intracellular transport is mainly through lysosome- or endoplasmic reticulum-mediated transport pathways. Uptake mechanism and transport pathway of RVGPR9-SSL and RVGP-R9-SSL in BMVEC cells Figure 3 As shown. RVGPR9-SSL enters BMVEC cells through receptor-mediated and caveolin-mediated mechanisms, and is mainly transported through the endoplasmic reticulum pathway, which can bypass lysosomes for transport, thereby effectively avoiding the influence of various hydrolases in lysosomes. RVGP-R9-SSL enters BMVEC cells through receptor-mediated and clathrin-mediated pathways, and is mainly transported through the lysosomal pathway, and has the ability to escape lysosomes. Combined with the integrity experiments after the two liposomes cross the BBB, it can be concluded that RVGPR9-SSL and RVGP-R9-SSL are eventually transported out of BMVEC cells in an intact form, and are expected to reach the lesions in the brain, such as gliomas, in an intact form to play a therapeutic role. In summary, both RVGPR9-SSL and RVGP-R9-SSL can bypass lysosomes or have the ability to escape lysosomes, and can cross the BBB in an intact form. Both are potential brain-targeted drug delivery systems. However, RVGPR9-SSL had a higher BBB permeability, indicating that it bypassed the lysosome and transported via the ER pathway with a higher efficiency of transport across the BBB.

[0063] Example 2

[0064] The transport mechanism of RVGPR9-SSL and RVGP-R9-SSL in C6 cells is studied as follows:

[0065] C6 cells were incubated with inhibitors (chlorpromazine, philipid and sodium azide) for 30 minutes. Subsequently, the incubated C6 cells were incubated with SSL (Cou-6), RVGPR9-SSL (Cou-6) or RVGP-R9-SSL (Cou-6) solutions containing inhibitors (preparation process is the same as in Example 1) for 1 hour. The test solution without inhibitors was used as the control group. Flow cytometry was used to detect the uptake of each group of solutions by C6 cells. The lysosomes of C6 cells were labeled with lyso-trackerRed, and the endoplasmic reticulum of C6 cells was labeled with the fluorescent probe ER-tracker-Red. The co-localization of each group of liposome solutions with the lysosomes and endoplasmic reticulum of C6 cells was consistent with the detection method in Example 1. Liposomes loaded with the chemotherapeutic drug doxorubicin (DOX) (SSL (DOX), RVGPR9-SSL (DOX), RVGP-R9-SSL (DOX)) were prepared according to the above method. The nuclei of C6 cells were labeled with the fluorescent probe Hoechst33342. The colocalization of SSL(DOX), RVGPR9-SSL(DOX) and RVGP-R9-SSL(DOX) with C6 cell nuclei was the same as in Example 1. We also used a ZetasizerNano ZS dynamic light scattering instrument to measure the Zeta potential in SSL(Cou-6), RVGPR9-SSL(Cou-6) or RVGP-R9-SSL(Cou-6) solutions (pH 7.4 and pH 6.5) containing inhibitors, running 20 times with 120 s of equilibrium each time. The results are shown in Table 1.

[0066] RVGPR9-SSL and RVGP-R9-SSL, two dual-mediated brain-targeted liposomes, have high blood-brain barrier permeability, especially RVGPR9-SSL, which has a 4h blood-brain barrier permeability of 10.31%, providing a possibility for the treatment of glioma. Tumor tissue has an acidic microenvironment with a pH value close to 6.5 (Xiang B, Dong D, Shi N, Gao W, Yang Z, Cui Y, Cao D, Qi XR*. PSA-responsive and PSMA-mediated multifunctional liposomes for targeted therapy of prostate cancer. Biomaterials. 2013, 34: 6976-6991.). Unlike BMVEC cells that express nAChR on the surface, glioma cells express M2 acetylcholine receptors on the surface (Ferretti M, Fabbiano C, Di Bari M, Conte C, Castigli E, Sciaccaluga M, Ponti D, Ruggieri P, Raco A, Ricordy R, Calogero A, Tata AM. M2 receptor activation inhibits cell cycle progression and survival in human glioblastoma cells [J]. Journal of Cellular and Molecular Medicine, 2013, 17 (4): 552-566.). Due to the acidic microenvironment of the tumor and the specificity of the surface receptors, the transport mechanism of RVGPR9-SSL and RVGP-R9-SSL in C6 cells may be different from that of BMVEC cells. Therefore, in order to further illustrate the potential therapeutic effects of the two dual-mediated brain-targeted liposomes on glioma, we investigated the uptake mechanism and transport pathway of RVGPR9-SSL and RVGP-R9-SSL in C6 cells. The results showed that SSL, RVGPR9-SSL and RVGP-R9-SSL were taken up by C6 cells in a clathrin-mediated manner. The uptake of the three liposomes was energy-dependent ( Figure 4 The results of the co-localization experiment between liposomes and lysosomes or endoplasmic reticulum in C6 cells showed that the co-localization rate between RVGPR9-SSL and RVGP-R9-SSL and lysosomes was significantly higher than that between ER and lysosomes, indicating that these two dual-mediated brain-targeted liposomes were mainly transported through the lysosomal pathway of C6 cells ( Figure 4Figures b-e in the middle, the data were processed using LAS X software and the Pearson coefficient was calculated). In addition, both RVGPR9-SSL and RVGP-R9-SSL showed lysosomal escape ability in C6 cells ( Figure 4 b-e). This may be related to the enhanced positive charge on the liposome surface in the acidic microenvironment of the tumor (Table 1). RVGPR9-SSL and RVGP-R9-SSL enter tumor cells mainly through the R9-mediated uptake mechanism in the acidic microenvironment, and are mainly transported through the lysosomal pathway in C6 cells.

[0067] Table 1 Zeta potential of Cou-6 loaded liposomes at pH 7.4 and pH 6.5 (n=3)

[0068] pH SSL(Cou-6) RVGPR9-SSL(Cou-6) RVGP-R9-SSL(Cou-6) 7.4 -7.47±0.16 -5.27±0.10 -5.68±0.13 6.5 1.54±0.12 6.77±0.31* 4.21±0.69*

[0069] Next, the chemotherapy drug doxorubicin (DOX) was encapsulated in SSL, RVGPR9-SSL, and RVGP-R9-SSL. The co-localization of SSL (DOX), RVGPR9-SSL (DOX), and RVGP-R9-SSL (DOX) with the nucleus of C6 cells was investigated. Figure 5 As shown, the co-localization rate of RVGPR9-SSL(DOX) and RVGP-R9-SSL(DOX) with the cell nucleus gradually increased at 1, 2, and 4 h after administration, indicating that the liposomes can aggregate to the cell nucleus region ( Figure 5 In addition, the co-localization rate of RVGPR9-SSL(DOX) and RVGP-R9-SSL(DOX) with the cell nucleus was significantly higher than that of SSL(DOX). This may be related to the lysosomal escape ability of the two dual-mediated brain-targeted liposomes, laying the foundation for them to reach the cell nucleus and exert their therapeutic effects ( Figure 5 Figure a, Figure b). In summary, RVGPR9-SSL and RVGP-R9-SSL are transported through the lysosomal pathway in C6 cells, both have the ability to escape from lysosomes, and can aggregate in the nucleus to exert therapeutic effects. Combined with the previous BBB permeability study results (Hei Yu, Mei Zi, Chen Yang, et al. Dual-mediated brain-targeted liposomes enhance the in vitro blood-cerebrospinal fluid barrier permeability of doxorubicin-loaded liposomes [J]. Journal of Medical Research, 2024, 53(01): 30-35.), RVGPR9-SSL may play a better role in the treatment of gliomas due to its higher BBB permeability.

[0070] Example 3

[0071] The uptake of RVGPR9-SSL and RVGP-R9-SSL in the BBB / C6 co-culture model was studied as follows:

[0072] Obtaining an in vitro BBB model: BMVEC cells in the culture flask that were 90% confluent were evenly digested with BMVEC-specific culture medium to form a single-cell suspension, and the cells were counted. Then, the BMVEC cells were plated at 1×10 5 The density of cells / well was inoculated into the Transwell donor pool, and the volume of the cell suspension added to the donor pool was 1.5mL. Then 2mL of BMVEC-specific culture medium was added to the Transwell recipient pool, and then the Transwell culture plate was transferred to a 5% CO2, 37°C incubator for culture. Culture in the incubator for 3-7 days until the BBB model was established. The culture medium was changed every 2 days during the culture process.

[0073] The composition of BMVEC-specific culture medium is as follows: DMEM culture medium with 20% fetal bovine serum (FBS), 40U / mL heparin, 100U / mL penicillin, 100ug / mL streptomycin, 2mmol / L L-glutamine, 100ug / mL endothelial cell growth factor (ECGF), and 2.32mg / mL Hepes buffer added.

[0074] 4h leakage experiment: Place the Transwell culture plate that has been cultured for 3-7 days under a microscope for observation. After the cells have merged, add BMVEC-specific culture medium to the donor pool of the Transwell culture plate so that the liquid level of the donor pool of the Transwell culture plate is 5mm higher than the liquid level of the receiving pool. Then place the Transwell culture plate in a 5% CO2, 37°C incubator for 4h. After that, take out the Transwell culture plate and observe whether the liquid level difference between the donor pool and the receiving pool can be maintained.

[0075] TEER measurement: Place the Transwell culture plate cultured for 3-7 days under a microscope for observation. After cell fusion, use an EVOM resistance meter to measure the resistance between the Transwell donor pool and the receiver pool, i.e., TEER. If TEER is greater than 200Ω·cm 2 , it means that the BBB model is successfully established.

[0076] After 4h leakage test and TEER identification, the Transwell culture plate was taken out, the culture medium of the donor pool was discarded, and the plate was washed three times with sterile PBS buffer, then fixed at 4°C for 2h with 2% glutaraldehyde fixative, the 2% glutaraldehyde was discarded, and the plate was washed three times with sterile PBS, then fixed at 4°C for 2h with 1% kaolin fixative, the 1% kaolin was discarded, and the plate was washed three times with sterile PBS buffer, dehydrated with 50% ethanol for 10min, and then dehydrated with 70% ethanol for 10min, and then gold sprayed. The tight junctions between cells on the polycarbonate membrane of the Transwell donor pool were observed with SEM to further determine the fusion state of BMVEC cells and evaluate whether the in vitro BBB model was successfully established.

[0077] Construction of BBB / C6 co-culture model: C6 cells were cultured at 1×10 5 The density of C6 cells was inoculated into a six-well culture plate. After the C6 cells grew uniformly on the wall, the donor pool of the in vitro BBB model was inserted above the six-well cell culture plate. C6 cells were co-cultured with BMVEC cells for 12 hours to construct a BBB / C6 co-culture model. Flow cytometry was used to detect the uptake of liposome solution in the BBB / C6 co-culture model. At the same time, a 4-hour leakage experiment and TEER measurement were performed on the constructed BBB / C6 co-culture model. Drug addition: RVGPR9-SSL and RVGP-R9-SSL were diluted with serum-free DMEM medium to a final phospholipid concentration of 0.2 mg / mL, and then the reserve solution was sterilized by a 0.22 μm sterile microporous filter membrane. Subsequently, 1.5 mL of the aforementioned diluted RVGPR9-SSL and RVGP-R9-SSL solutions were added to the donor pool of the BBB / C6 co-culture model, and the PBS well was used as a blank control. Each sample was repeated three times. The six-well plate was then placed in a 37°C constant temperature incubator for 4 hours.

[0078] In this study, an in vitro BBB model was first constructed and tested by a 4-h leakage test (the liquid level difference could be maintained at 0.5 cm) and by measuring the transmembrane resistance (greater than 200 Ω·cm 2 ), confirming that the in vitro BBB model was successfully constructed. Based on the successful construction of the BBB model, the BBB / C6 co-culture model was constructed ( Figure 6 Figure a). The 4-hour leakage test showed that the height difference of the liquid in each well of each BBB / C6 co-culture model could be maintained at 0.5 cm. The TEER values ​​were all greater than 200 Ω·cm 2 Then, the cell status of each group of BBB / C6 co-culture model was observed by SEM ( Figure 6(Fig. b), the results show that BMVEC cells are densely arranged and there are obvious tight junctions between cells. Therefore, C6 cells co-cultured with the BBB will not destroy the integrity of the BBB. This BBB / C6 co-culture model can be used to study the ability of RVGPR9-SSL and RVGP-R9-SSL to be taken up by C6 cells after crossing the BBB. The uptake results of RVGPR9-SSL and RVGP-R9-SSL in the BBB / C6 co-culture model can reflect the potential of the two liposomes to deliver drugs to gliomas.

[0079] Figure 6 The results showed that the average uptake of RVGPR9-SSL in the BBB / C6 co-culture model was significantly higher than that of RVGP-R9-SSL, indicating that RVGP-R9-SSL has better glioma targeting potential ( Figure 6 This may be related to the higher BBB permeability of RVGPR9-SSL. Within the same incubation time, RVGPR9-SSL enters the brain at a higher concentration and thus reaches a higher concentration in the glioma area, thereby promoting the uptake of C6 cells.

[0080] In summary, the present invention conducted a comparative study on two dual-mediated brain-targeted liposomes RVGPR9-SSL and RVGP-R9-SSL constructed in the early stage. Their transport pathways, cell internalization mechanisms, and transport pathways across the BBBs were studied. The results show that different modification patterns affect the cell internalization mechanism and transport pathway of dual-mediated brain-targeted liposomes, thereby affecting their BBB permeability and brain targeting efficiency. RVGPR9-SSL can be effectively transported across the BBB, has good brain targeting ability, can bypass lysosomal transport and target tumor cell nuclei. Therefore, RVGPR9-SSL is an ideal brain-targeted drug delivery system, which is expected to play a good therapeutic role in gliomas. In addition, the research results of the present invention provide important guidance and data support for the construction of subsequent dual-mediated brain-targeted liposomes.

[0081] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for evaluating the therapeutic effect of brain glioma based on a dual-mediated liposome drug delivery system, characterized in that: The steps include: Step 1, obtaining a dual-mediated liposome drug delivery system; Step 2, respectively evaluating the transport mechanism of the dual-mediated liposome drug delivery system in BMVEC cells and C6 cells; Step 3, obtaining a BBB / C6 co-culture model, and evaluating the transport of the dual-mediated liposome drug delivery system in the BBB / C6 co-culture model; Step 4: Based on the results of step 2 and step 3, the evaluation results of the dual-mediated liposome drug delivery system for the treatment of brain glioma are obtained.

2. The evaluation method according to claim 1, characterized in that: In the step 1, the dual-mediated liposome drug delivery system is RVGPR9-SSL and RVGP-R9-SSL.

3. The evaluation method according to claim 2, characterized in that: In the step 2, chlorpromazine is used as a clathrin inhibitor, filipin is used as a caveolae inhibitor, RVG29 is used as an AChR inhibitor, and sodium azide is used as an energy inhibitor to evaluate the transport mechanism of the dual-mediated liposome drug delivery system in BMVEC cells and C6 cells.

4. The evaluation method according to claim 3, characterized in that: The step 2 comprises the following steps: Step 2-1, using DMEM medium without phenol red to prepare inhibitor reserve solutions of chlorpromazine, philipin, sodium azide, and RVG29 respectively; and using DMEM medium without phenol red to prepare coumarin-encapsulated SSL, RVGPR9-SSL, and RVGP-R9-SSL liposome test solutions respectively; the four inhibitor reserve solutions and the three test solutions are mixed respectively to form 12 groups of inhibitor-containing liposome solutions; Step 2-2, incubating BMVEC cells or C6 cells with chlorpromazine, philipid, sodium azide and RVG29 respectively, and then incubating the BMVEC cells or C6 cells after the incubation with a liposome solution containing an inhibitor, the culture solution after the incubation is used as the test group, and the test solution without the inhibitor is used as the control group, and the uptake of each group of experimental solutions by BMVEC cells or C6 cells is detected by flow cytometry; Step 2-3, using the fluorescent probe lyso-tracker Red to label the lysosomes of BMVEC cells, and using the fluorescent probe ER-tracker-Red to label the endoplasmic reticulum of BMVEC cells; then incubating the coumarin-loaded SSL, R9-SSL, RVGP-SSL, RVGPR9-SSL and RVGP-R9-SSL with the BMVEC cells labeled with the fluorescent probes, and observing the colocalization of each liposome solution with the lysosomes and endoplasmic reticulum of the BMVEC cells by laser confocal microscopy; Step 2-4, using the fluorescent probe lyso-tracker Red to label the lysosomes of C6 cells, and using the fluorescent probe ER-tracker-Red to label the endoplasmic reticulum of C6 cells; then incubating SSL, RVGPR9-SSL and RVGP-R9-SSL loaded with doxorubicin with the C6 cells labeled with the fluorescent probes, and observing the co-localization of each liposome solution with the lysosomes and endoplasmic reticulum of C6 cells by laser confocal microscopy; Step 2-5, measuring the Zeta potential of the liposome solution containing the inhibitor at pH 7.4 and pH 6.

5.

5. The evaluation method according to any one of claims 1 to 4, characterized in that: The step 3 comprises the following steps: Step 3-1, obtaining an in vitro BBB model; Step 3-2, co-culturing C6 cells with the in vitro BBB model to obtain a BBB / C6 co-culture model; Step 3-3, adding the dual-mediated liposome drug delivery system to the BBB / C6 co-culture model, and then evaluating the uptake results of the dual-mediated liposome drug delivery system in the BBB / C6 co-culture model.

6. A kit for evaluating the therapeutic effect of glioma based on a dual-mediated liposome drug delivery system, characterized in that: include: BMVEC cell evaluation unit; C6 Cell Evaluation Unit; BBB / C6 co-culture model evaluation unit.

7. The kit according to claim 6, characterized in that The BMVEC cell evaluation unit comprises: BMVEC cells, a reserve inhibitor solution, a fluorescent dye for marking a dual-mediated liposome drug delivery system, and a fluorescent probe for marking BMVEC cells.

8. The kit according to claim 6, characterized in that The C6 cell evaluation unit comprises: C6 cells, a reserve inhibitor solution, a fluorescent dye for marking a dual-mediated liposome drug delivery system, a fluorescent probe for marking C6 cells, and an anti-tumor drug; preferably, the anti-tumor drug is doxorubicin.

9. The kit according to claim 6, characterized in that The BBB / C6 co-culture model evaluation unit includes: a BBB / C6 co-culture model.

10. The kit according to claim 7 or 8, characterized in that The preparation method of the inhibitor reserve solution is as follows: the inhibitors chlorpromazine, philipin, sodium azide and RVG29 are respectively prepared into the inhibitor reserve solution using a DMEM culture medium without phenol red.

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