CN106333926A - Brain tumor multiple targeting drug delivery system of stability polypeptide mediated cross-barrier film - Google Patents

Abstract

The invention belongs to the field of pharmacy, and relates to a brain tumor targeting carrier system mediated by stable polypeptides across multiple barrier membranes, which is composed of stable polypeptides ^D CDX and c(RGDyK) and liposomes. The drug delivery system is capable of resisting Enzyme barrier in vivo, brain tumor multiple targeting liposome drug delivery system mediated across the blood-brain barrier (BBB) and blood-brain tumor barrier (BBTB). Experimental results show that the liposome can deliver the encapsulated model drug to the target tissue, such as crossing the BBB in vitro and entering the tumor sphere in the lower chamber, and entering glioma in vivo, and the encapsulation of doxorubicin can prolong the life of the glioma model. The survival period of animals is significantly improved. The multi-targeted liposome drug delivery system modified by the stable polypeptide has a good application prospect in the diagnosis and treatment of brain tumors.

Description

A stable polypeptide-mediated multi-targeted drug delivery system for brain tumors across barrier membranes

technical field

The invention belongs to the field of pharmacy, and relates to a stable polypeptide-modified liposome carrier system for multiple targeted drug delivery, in particular to a stable polypeptide ^D CDX whose amino acid sequence is G ^D R ^D E ^D I ^D R ^D TG ^D R ^D A ^D E ^D R ^D W ^D S ^D E ^D K ^D F and c(RGDyK) Co-modified Brain Tumor Targeting Liposome Carriers Against In vivo Enzyme Barrier, Crossing Blood-Brain Barrier and Blood-Brain Tumor Barrier System, preparation method and application thereof in the preparation of drugs for the diagnosis and treatment of brain tumors.

Background technique

Studies have reported that in current clinical practice, the treatment of brain tumors is mainly surgical resection, supplemented by radiotherapy and chemotherapy. The five-year survival rate of patients is low, and brain tumors seriously threaten human health and life. Studies have shown that the brain, as the center of the human body, has many forbidden areas. It is difficult to remove the tumor completely by surgery, and the residual tumor cells are very easy to relapse. Therefore, postoperative radiotherapy and chemotherapy are particularly important; It is also difficult to enter the brain and limit its efficacy. Therefore, the targeted delivery of chemotherapy drugs for brain tumors has become a hot spot in this technical field.

In recent years, the research on targeted drug delivery system has made some progress, but there are still many problems in the targeted therapy of brain tumors due to the special environment of the brain. In the early stage of tumor development, tumor cells supply nutrients through brain capillaries, and the blood-brain barrier (BBB) is complete. BBB is a structure, function and enzyme barrier composed of brain microvascular endothelial cells, astrocytes and pericytes. It can selectively deliver nutrients and essential endogenous substances into the brain, and discharge toxic metabolites and exogenous substances into the brain to maintain the stability of the brain's internal environment. Practice has shown that the existence of the BBB makes it difficult for almost all macromolecules and about 98% of small molecule drugs to enter the brain, and only molecules with extremely small molecular weight (<400-500 Daltons), fat-soluble and electrically neutral can undergo passive diffusion across the BBB. With the development of brain tumors, tumor neovascularization can lead to BBB destruction, but it is different from peripheral tumors. Brain tumor neovascularization is relatively dense and poorly permeable. There is a blood-brain tumor barrier (BBTB), but some tumor cells Intact BBB is still present in the infiltrated area. With the further deterioration of the brain tumor, the EPR effect appears in the tumor site, but its effect is obviously weaker than that of the peripheral solid tumor. The gap of the new blood vessel in the human glioma is only 7-100nm. Barrier membranes that exist at different stages of brain tumor development limit the drug delivery system from reaching the lesion site.

In view of the expression of multiple receptors on the BBB, including low-density lipoprotein receptors, transferrin receptors, and nicotinic acetylcholine receptors, the polypeptide ligands in the blood circulation and their modified drug delivery systems can be receptor-mediated The endocytotic transport mode crosses the BBB. Considering the influence of the in vivo environment, especially the enzyme barrier of serum and BBB, on the stability of the targeting ligand, which may reduce the targeting efficiency of the drug delivery system, and the D-configuration polypeptide has the advantage of good resistance to enzymatic degradation, the present application The inventor of the previous work designed and synthesized a stable brain-targeting polypeptide molecule ^D CDX, which has a high affinity with nicotinic acetylcholine receptors, and can carry the drug delivery system to penetrate the blood-brain barrier into the brain. Studies have shown that integrins are highly expressed on both BBTB and tumor cells, and peptides containing arginine, glycine and aspartic acid (RGD) sequences can specifically bind to integrins, among which cyclic peptide c (RGDyK) has a higher High stability can effectively mediate the drug delivery system to target BBTB and tumor cells.

Therefore, the inventors of the present application intend to aim at the barrier membranes existing in different stages of brain tumor development, considering that the enzyme environment in the body affects the stability of the targeting polypeptide molecule and may reduce the targeting efficiency of its mediated drug delivery system, and provides a stable A drug delivery system for brain tumors that crosses multiple barrier membranes co-modified by the sex polypeptide molecule ^D CDX and c(RGDyK), utilizes the characteristics of multiple targeting and crossing multiple barrier membranes of this targeted drug delivery system to realize the Targeted delivery of tumor diagnostic or therapeutic drugs.

Contents of the invention

The purpose of the present invention is to aim at the barrier membranes existing in different stages of brain tumor development, considering that the enzyme environment in vivo affects the stability of targeting polypeptide molecules and may reduce the targeting efficiency of its mediated drug delivery system, and provides a stable polypeptide modified A liposome carrier system for multiple targeted drug delivery, the targeted drug delivery system is a brain tumor targeted drug delivery system that is co-modified by stable polypeptide molecules ^D CDX and c(RGDyK) across multiple barrier membranes, the targeted drug delivery system In the drug delivery system, the stable polypeptide ^D CDX and c(RGDyK) are jointly modified to resist the enzyme barrier in vivo and mediate multiple barrier membranes across the BBB and BBTB, which can realize the targeted diagnosis and treatment of brain tumors.

The present invention utilizes the characteristics of multiple targeting and crossing multiple barrier membranes of the targeted drug delivery system to realize the targeted delivery of drugs for the diagnosis or treatment of brain tumors at various stages; The drug delivery system uses the specific combination of the first-level targeting molecule ^D CDX and the nicotinic acetylcholine receptor on the BBB to mediate the drug delivery system across the BBB, and integrates with the tumor cells through the second-level targeting molecule c (RGDyK) The combination of c(RGDyK) and the integrin on the neovascularization of the brain tumor can specifically bind to the integrin on the neovascularization of the brain tumor to cross the BTBTB, and Targeted killing of tumor cells; by controlling the particle size of the drug delivery system to be less than 100nm, the weak EPR effect can be used to reach the brain tumor site, and c(RGDyK) can be used to mediate targeted killing of tumor cells.

Concretely, a liposome carrier system for stabilizing polypeptide-modified multiple targeted drug delivery of the present invention is characterized in that it consists of stabilizing polypeptides ^D CDX and c(RGDyK) and liposomes, said ^D CDX The amino acid sequence is G ^D R ^D E ^D I ^D R ^D TG ^D R ^D A ^D E ^D R ^D W ^D S ^D E ^D K ^D F.

The liposome drug delivery system co-modified by ^D CDX and c(RGDyK) of the present invention can include doxorubicin, epirubicin, paclitaxel, docetaxel, camptothecin, hydroxycamptothecin, 9-nitro Camptothecin, vincristine, bortezomib, carfenazomib, methotrexate, cisplatin and other small molecule anti-tumor drugs; can also contain TRAIL protein, ^L PMI, ^D PMI, TRAIL gene, siRNA, etc. Biological macromolecular anti-tumor drugs can also carry diagnostic drugs such as fluorescein isothiocyanate FITC, near-infrared dyes Cy5.5, Cy7, IR820, DiR, and magnetic resonance imaging agent Gd.

The present invention demonstrates the in vitro serum stability and liver lysosome homogenate stability of ^D CDX and c(RGDyK).

In the present invention, the membrane material of the liposome carrier system includes the following five components: a: natural phospholipids or synthetic phospholipids, and b: cholesterol, and c: methoxypolyethylene glycol-phospholipid complex (methoxy The molecular weight of polyethylene glycol is 1000-5000), and d: polypeptide-polyethylene glycol-phospholipid complex containing ^D CDX sequence polypeptide (the molecular weight of polyethylene glycol is 1000-8000) and e: containing c (RGDyK ) sequence polypeptide polypeptide-polyethylene glycol-phospholipid complex (the molecular weight of polyethylene glycol is 1000～8000); the molar ratio between each component is a:b=5:1～1:2, a:c =1000:1～1000:100, a:d=1000:1～1000:100, a:e=1000:1～1000:100.

Described natural phospholipid or synthetic phospholipid is selected from lecithin, soybean lecithin, hydrogenated soybean lecithin, dipalmitoyl phosphatidyl choline, dimyristoyl phosphatidyl choline, distearoyl phosphatidyl choline, hydrogenated egg phosphatidyl choline Alkaline, yolk sphingomyelin, palmitoyl oleoyl phosphatidylcholine;

The methoxypolyethylene glycol-phospholipid complex, the polypeptide-polyethylene glycol-phospholipid complex containing the ^D CDX sequence polypeptide and the polypeptide-polyethylene glycol-phospholipid complex containing the c(RGDyK) sequence polypeptide The phospholipids in can be distearoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, hydrogenated soybean phosphatidylethanolamine, hydrogenated egg phosphatidylethanolamine, soybean phosphatidylethanolamine or egg phosphatidylethanolamine;

In the present invention, the molecular weight of methoxypolyethylene glycol in the methoxypolyethylene glycol-phospholipid complex is 1000 to 5000, and the polyethylene glycol in the polypeptide-polyethylene glycol-phospholipid complex containing ^D CDX sequence polypeptide The molecular weight is 1000-8000, and the molecular weight of polyethylene glycol in the polypeptide-polyethylene glycol-phospholipid complex containing c(RGDyK) sequence polypeptide is 1000-8000;

The methoxypolyethylene glycol-phospholipid complex and the polypeptide-polyethylene glycol-phospholipid complex containing the ^D CDX sequence polypeptide and the polypeptide-polyethylene glycol-phospholipid complex containing the c(RGDyK) sequence polypeptide The phospholipid in is selected from distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, hydrogenated soybean phosphatidylethanolamine, hydrogenated egg phosphatidylethanolamine;

In the present invention, the polypeptide-polyethylene glycol-phospholipid complex containing the ^D CDX sequence polypeptide or the polypeptide-polyethylene glycol-phospholipid complex containing the c(RGDyK) sequence polypeptide is synthesized by covalently bonding with polyethylene glycol Alcohol-phospholipids are linked into complexes: ^D CDX or c(RGDyK) sequence polypeptides provide a free sulfhydryl group, polyethylene glycol-phospholipids provide a maleimide group, and the two are linked by the maleimide group and sulfhydryl group Michael addition reaction linking into covalent complexes;

In the present invention, liposomes are prepared by rotary evaporation-film hydration-extrusion method, a certain proportion of a, b, c, d and e are dissolved in chloroform, and ^D CDX and c are prepared by rotary evaporation-film hydration method (RGDyK) co-modified liposomes ( ^D CDX/c(RGDyK)-LS), reduce the liposome particle size by extruding through the membrane to obtain liposomes; the laser light scattering method measures the particle size distribution, and its The average particle diameter is 50-500nm.

In the present invention, brain capillary endothelial cell line (bEnd.3 cell), primary brain capillary endothelial cell and glioma cell (U87 cell) are carried out to ^D CDX/c(RGDyK)-LS/FAM, ^D CDX - The uptake experiments of LS/FAM, c(RGDyK)-LS/FAM and mPEG-liposome/FAM, the results confirmed that ^D CDX/c(RGDyK)-LS has both in vitro brain targeting ability and in vitro tumor targeting ability.

The present invention constructs a BBB in vitro model and investigates the ability of CDX/c(RGDyK)-LS/FAM to transport through the BBB, and the results confirm that ^D CDX/c(RGDyK)-LS has the ability to cross the blood-brain barrier in vitro.

The present invention constructed an in vitro model of BBTB and investigated the ability of CDX/c(RGDyK)-LS/FAM to transport through BBTB. The results confirmed that ^D CDX/c(RGDyK)-LS has the ability to cross the blood-brain tumor barrier in vitro.

In the present invention, the co-culture model of BBB and U87 tumor spheres was used to investigate the uptake of ^D CDX/c(RGDyK)-LS/FAM tumor spheres. The results confirmed that ^D CDX/c(RGDyK)-LS has the ability to cross the BBB barrier in vitro and brain tumor targeting ability.

The present invention develops ^D CDX/c(RGDyK)-LS/DiR, ^D CDX-LS/DiR, c(RGDyK)-LS/DiR and mPEG-liposome/DiR labeled with near-infrared dye DiR in different gliomas Each stage was injected into nude mice bearing orthotopic glioma model through the tail vein, and observed under the in vivo imager. The results proved that ^D CDX/c(RGDyK)-LS can target brain tumors in vivo.

In the present invention, the orthotopic brain glioma animal model was injected with ^D CDX/c(RGDyK)-LS/DOX, ^D CDX-LS/DOX, c(RGDyK)-LS/DOX, LS/DOX, free A The in vivo anti-tumor effect of different doxorubicin-loaded drug delivery systems was evaluated using the survival time as an index. The results proved that ^D CDX/c(RGDyK)-LS has anti-brain tumor ability in vivo.

Experimental research results show that the stable polypeptide ^D CDX and c(RGDyK) involved in the present invention are jointly modified to resist the enzyme barrier in vivo and mediate the glioma-targeted drug delivery system across BBB and BBTB multiple barrier membranes, which can be used to prepare Drug delivery vector system for targeted diagnosis and therapy of brain tumors.

Description of drawings

Figure 1. Serum stability of ^D CDX and c(RGDyK),

It shows the stability of ^D CDX and c(RGDyK) in 50% rat serum, and both ^D CDX and c(RGDyK) have high serum stability.

Figure 2. Hepatic lysosomal enzyme stability of ^D CDX and c(RGDyK),

The stability of ^D CDX and c(RGDyK) in liver lysosome homogenate is shown, and both ^D CDX and c(RGDyK) have high stability of lysosomal enzymes.

Figure 3, the ¹ H-NMR spectrum of ^D CDX-PEG ₃₄₀₀ -DSPE and c(RGDyK)-PEG ₃₄₀₀ -DSPE,

It shows that the NMR spectrum of Mal-PEG ₃₄₀₀ -DSPE shows a maleimide peak at 6.7ppm, while the peak disappears in the NMR spectra of ^D CDX-PEG ₃₄₀₀ -DSPE and c(RGDyK)-PEG ₃₄₀₀ -DSPE, It shows that the maleimide group in Mal-PEG ₃₄₀₀ -DSPE has reacted with ^D CDX or c(RGDyK).

Figure 4. Uptake of 5-FAM-loaded liposomes by brain capillary endothelial cells and U87 cells,

Among them, Figures A and C are the laser confocal photos and flow cytometry results of the liposomes loaded with 5-FAM and the primary brain capillary endothelial cells at 37°C for 12 hours respectively. The uptake of DCDX-LS and DCDX/c(RGDyK)-LS was significantly higher than that of c(RGDyK)-LS and non-target liposomes, and the uptake percentages of the former two had no significant difference;

Figures B and D are laser confocal photos and flow cytometry results of the liposomes loaded with 5-FAM and U87 cells for 4 hours at 37°C, respectively. The uptake of (RGDyK)-LS was significantly higher than that of DCDX-LS and target-free liposomes, and there was no significant difference in the uptake percentages of the former two.

Figure 5. BBB transport of liposomes in vitro

The figure shows the percentage of each liposome transported across the blood-brain barrier model in vitro at different time points. It can be seen from the figure that at 30min, 1h, 2h and 4h, the amount of ^D CDX-LS and ^D CDX/c(RGDyK)-LS transported through the in vitro BBB model was significantly higher than that of c(RGDyK)-LS and non-target liposomes, And there was no significant difference in the percentage of transfer between the former two.

Figure 6. BBBTB transport of liposomes in vitro,

It shows the percentage of each liposome transported across the in vitro blood-brain tumor barrier model at different time points. At 30min, 1h, 2h and 4h, c(RGDyK)-LS and ^D CDX/c(RGDyK)-LS transported through the in vitro The amount of BBTB model was significantly higher than that of ^D CDX-LS and non-target liposomes, and the transfer percentages of the former two had no significant difference.

Figure 7. Lysosome co-localization during liposome BBB transport in vitro,

It shows the lysosome co-localization of ^D CDX/c(RGDyK)-LS/FAM during in vitro blood-brain barrier transport, and the liposome has good co-localization with lysosome in in vitro blood-brain barrier.

Figure 8. Liposome U87 three-dimensional tumorsphere uptake,

It shows the uptake of liposome serum by U87 three-dimensional tumor spheres in the transwell lower chamber before and after crossing the in vitro blood-brain barrier model before and after incubation. Only liposomes co-modified by ^D CDX and c(RGDyK) can effectively cross the blood-brain barrier and be absorbed by the lower chamber. U87 three-dimensional tumorsphere uptake, and its uptake did not change significantly before and after serum incubation.

Figure 9. The distribution in the brain of liposomes loaded with near-infrared dyes,

It shows that nude mice bearing orthotopic glioma were injected with ^D CDX/c(RGDyK)-LS/DiR, ^D CDX-LS/DiR, c (RGDyK)-LS/DiR and LS/DiR 12 hours later in vitro brain imaging distribution results, compared with other groups of liposomes, ^D CDX/c(RGDyK)-LS showed the highest distribution in the brain tumor area.

Figure 10. Characterization of doxorubicin-loaded liposomes,

It shows that the particle diameters of the four kinds of doxorubicin-loaded liposomes are all about 90nm, the particles are round, and there is no significant difference in size and shape.

Figure 11, the in vitro drug effect of liposomes loaded with doxorubicin on U87 cells,

The anti- ^U87 cell activity curves of free doxorubicin and ^doxorubicin -loaded liposomes in vitro are shown: After c(RGDyK)-LS/DOX and free doxorubicin were incubated for 4 hours and then replaced with fresh culture medium for 72 hours, the IC ₅₀ were 83.1, 75.8, 13.2, 12.0, 0.5 μM, and the four liposomes all had the ability to inhibit In vitro growth of U87 cells, the in vitro antitumor activity of liposomes containing c(RGDyK) targeting molecules (including c(RGDyK)-LS/DOX and ^D CDX/c(RGDyK)-LS/DOX) was significantly greater than that of LS/ DOX and ^D CDX-LS/DOX have similar antitumor activity in vitro.

Figure 12. Survival curve of nude mice with glioma orthotopic tumor model,

Among them, the average survival time of normal saline group, free doxorubicin group, LS/DOX group, ^D CDX-LS/DOX group, c(RGDyK)-LS/DOX group and ^D CDX/c(RGDyK)-LS/DOX group They were 26, 28, 29, 30.5, 32.5 and 36.5 days respectively. Compared with other groups, ^D CDX/c(RGDyK)-LS/DOX could significantly prolong the survival time of nude mice with glioma orthotopic tumor model.

detailed description

The following examples will help to further understand the present invention, but the present invention is not limited to the scope of the following description.

Example 1D Serum Stability of CDX and c( ^RGDyK )

Make ^D CDX or c(RGDyK) into a 1 mg/mL solution with PBS, take 0.1 mL and add it to 0.9 mL of 50% rat serum, incubate at 37°C, take out 100 μL of the reaction solution at each time point, add acetonitrile (containing 0.1 %TFA) to precipitate proteins in serum, let stand at 4°C for 10 min, centrifuge at 12000 r/min for 10 min, and take 20 μL of supernatant for qualitative and quantitative analysis by HPLC, the results are shown in Figure 1.

Example 2 Liver lysosomal enzyme stability of ^D CDX and c(RGDyK)

Take fresh rat liver, add 0.3M ice sucrose solution to homogenate, centrifuge at 700×g for 10min to remove cell debris, combine the supernatant and centrifuge again, collect the supernatant and centrifuge at 10000×g for 10min, collect the precipitate and add 20mL of 0.3M sucrose Mix the solution (containing 1mM CaCl ₂ ), incubate at 37°C for 5min, add 20mL of 50% Percoll, centrifuge at 10000×g for 10min, collect the precipitate, resuspend with 0.3M sucrose solution, and centrifuge at 10000×g for 10min. The precipitate was collected, washed twice, and finally the liver lysosome homogenate was resuspended with 0.3M sucrose solution, aliquoted, and stored at -80°C for future use. The protein content was determined with a BCA kit (using BSA as a standard protein to make a standard curve);

Add 100 μL of 1 mg/mL liver lysosome homogenate and 100 μL of 1 mg/mL CDX or c(RGDyK) solution to 800 μL of 0.2M sodium acetate buffer at pH 5.0 (hepatic lysosome homogenate: polypeptide=1: 1, w/w). After incubation at 37°C for 2 min and 15 min, 100 μL of the reaction solution was taken, and 20 μL of 15% trichloroacetic acid (TCA) was added to terminate the reaction. After centrifugation at 12000 r/min for 10 min, 20 μL of the supernatant was taken for HPLC analysis, and the results are shown in Figure 2.

Example 3 Synthesis and Characterization of ^D CDX-PEG ₃₄₀₀ -DSPE and c(RGDyK)-PEG ₃₄₀₀ -DSPE

Dissolve ^D CDX-Cys in 0.1M PBS solution (pH 7.2), take Mal-PEG ₃₄₀₀ -DSPE and dissolve it in DMF, mix the two and then react with magnetic stirring, monitor by HPLC, after the reaction of Mal-PEG-DSPE is complete Stop the reaction, remove excess ^D CDX-Cys and DMF by dialysis (molecular weight cut-off 3.5kDa), freeze-dry to obtain ^D CDX-PEG ₃₄₀₀ -DSPE, and characterize its structure by NMR. c(RGDyK)-Cys was reacted with Mal-PEG ₃₄₀₀ -DSPE according to the above method to obtain c(RGDyK)-PEG ₃₄₀₀ -DSPE, and its structure was characterized by NMR (as shown in FIG. 3 ).

Example 4 ^D In vitro targeting verification of CDX/c(RGDyK)-LS

Preparation of ^D CDX/c(RGDyK)-LS/FAM:

PEG-liposome membrane material formulation is HSPC/Chol/mPEG2000-DSPE (52:43:5, mol/mol), DCDX modified PEG liposome membrane material formulation is HSPC/Chol/mPEG2000-DSPE/DCDX- PEG3400-DSPE (52:43:3:2, mol/mol), the PEG liposome membrane material prescription of c (RGDyK) modification is HSPC/Chol/mPEG2000-DSPE/c (RGDyK)-PEG3400-DSPE (52: 43:4:1, mol/mol), DCDX and c(RGDyK) co-modified PEG liposome membrane material formulation is HSPC/Chol/mPEG2000-DSPE/DCDX-PEG3400-DSPE/c(RGDyK)-PEG3400-DSPE (52:43:2:2:1, mol/mol). The above-mentioned membrane material was weighed and dissolved in chloroform, and the organic solvent was removed by rotary evaporation under reduced pressure to obtain a uniform lipid membrane, which was dried in vacuum for 24 hours. Add 5-FAM aqueous solution for hydration, and shake in a water bath at 60°C for 2 hours to obtain a liposome suspension. In a water bath at 60°C, use a high-pressure homogenizer (if the liposome volume is less than 10 mL, use a micro extruder) to sequentially squeeze the liposomes through 400, 200, 100 and 50 nm nuclear pore membranes to make the particle size diameter decreases. Then use physiological saline as the eluent to separate and remove the unencapsulated 5-FAM through a Sephadex G-50 column to obtain liposomes loaded with 5-FAM;

In Vitro Targeting Validation of DCDX/c(RGDyK)-LS on Primary Brain Capillary Endothelial Cells

The brains of 4-week-old SD rats were decapitated, and the cerebral cortex was quickly separated in ice-cold D-Hanks solution. After removing the meninges and large blood vessels in the brain, they were cut into pieces and digested with collagenase and DNase at 37°C for 90 minutes. Then, 1000 Centrifuge at rpm for 8min, discard the supernatant, transfer to 20% BSA, centrifuge at 1000g/min at 4°C for 20min, discard the middle and upper layer liquid, transfer the bottom microvessels to culture medium, centrifuge at 1000rpm for 5min, and use DMEM culture solution containing 20% fetal bovine serum was made into microvascular segment suspension, inoculated in confocal dishes or 12-well plates, cultured at 37°C, 5% CO2 and saturated humidity for 24 hours, and replaced with endothelial cells containing puromycin After continuing to culture in the special culture medium for 72 hours, replace it with the special culture medium containing cell growth factors for endothelial culture for 72 hours to obtain primary brain capillary endothelial cells;

LS/FAM, DCDX-LS/FAM, c(RGDyK)-LS/FAM and DCDX/c(RGDyK)-LS/FAM solutions with a fluorescence concentration of 5 μM were prepared in DMEM culture solution containing 10% FBS, and the confocal glass Aspirate the culture solution in the bottom culture dish or 12-well plate, add the above solutions respectively, incubate at 37°C for 12 hours, and discard the liposome solution. After washing twice with PBS solution, the cells were fixed with formaldehyde, and the nuclei were stained with DAPI for 10 min. After washing twice with PBS, the cells were observed under a confocal microscope. See Figure 4A for photos of cell internalization. Or wash the plate twice with PBS, add trypsin to digest the cells, disperse the cells with the culture medium and centrifuge, discard the supernatant, wash twice with PBS, and finally disperse the cells in each well in 400 μL PBS, and measure the cells by flow cytometry, the results are shown in the figure As shown in 4C;

Validation of DCDX/c(RGDyK)-LS targeting U87 cells in vitro

Take the U87 cells in the logarithmic growth phase, digest the monolayer culture cells with 0.25% trypsin, make a single cell suspension with DMEM culture medium containing 10% fetal bovine serum, and inoculate 1×105 cells per well in the confocal Incubate in dish or 12-well culture plate at 37°C, 5% CO2 and saturated humidity for 24h. LS/FAM, DCDX-LS/FAM, c(RGDyK)-LS/FAM and DCDX/c(RGDyK)-LS/FAM solutions with a fluorescence concentration of 5 μM were prepared in DMEM culture solution containing 10% FBS, and the confocal glass Aspirate the culture solution in the bottom culture dish or 12-well plate, add the above solutions respectively, incubate at 37°C for 12 hours, and discard the liposome solution. After washing twice with PBS solution, the cells were fixed with formaldehyde, and the nuclei were stained with DAPI for 10 min. After washing twice with PBS, the cells were observed under a confocal microscope. See Figure 4B for photos of cell internalization. Or wash the plate twice with PBS, add trypsin to digest the cells, disperse the cells with the culture medium and centrifuge, discard the supernatant, wash twice with PBS, and finally disperse the cells in each well in 400 μL PBS, and measure the cells by flow cytometry, the results are shown in the figure 4D shown;

Study on the ability of DCDX/c(RGDyK)-LS to cross the blood-brain barrier in vitro

The brains of 4-week-old SD rats were decapitated, and the cerebral cortex was quickly separated in ice-cold D-Hanks solution. After removing the meninges and large blood vessels in the brain, they were cut into pieces and digested with collagenase and DNase at 37°C for 90 minutes. Then, 1000 Centrifuge at rpm for 8min, discard the supernatant, transfer to 20% BSA, centrifuge at 1000g/min at 4°C for 20min, discard the middle and upper layer liquid, transfer the bottom microvessels to culture medium, centrifuge at 1000rpm for 5min, and use DMEM culture medium containing 20% fetal bovine serum was made into microvascular segment suspension, inoculated in 24-well transwells pre-coated with rat tail collagen, and moved the transwells into a carbon dioxide incubator at 37°C, 5% CO2 and saturated humidity conditions Cultivate for 24 hours, change to endothelial-specific culture medium containing puromycin and continue to culture for 72 hours, then change to endothelial-specific culture medium containing cell growth factors and culture for 72 hours, the measured resistance exceeds 200Ω·cm2, that is, the in vitro BBB model is successfully established;

Prepare LS/FAM, DCDX-LS/FAM, c(RGDyK)-LS/FAM and DCDX/c(RGDyK)-LS/FAM solutions with a fluorescence concentration of 50 μM in DMEM culture solution containing 10% FBS, and place the transwell upper chamber Aspirate the culture solution in the cell, add the above solution respectively, take off the culture solution in the lower chamber at 30min, 1h, 2h and 4h to measure its fluorescence concentration, and the BBB transport results of each liposome are shown in Figure 5;

Study on the ability of DCDX/c(RGDyK)-LS to cross the blood-brain tumor barrier in vitro

The umbilical vein endothelial cells HUVECs and U87 were plated in the upper and lower chambers of the transwell at a ratio of 1:5, cultured for 72 hours, and LS/FAM with a fluorescence concentration of 50 μM was prepared in DMEM medium containing 10% FBS, DCDX-LS/ For FAM, c(RGDyK)-LS/FAM and DCDX/c(RGDyK)-LS/FAM solutions, suck out the culture solution in the upper chamber of the transwell, add the above solutions respectively, and remove the chamber at 30min, 1h, 2h and 4h The fluorescence concentration was measured in the culture solution, and the transport results of each liposome BTBB are shown in Figure 6;

In Vitro Brain Tumor Targeting Validation of DCDX/c(RGDyK)-LS

Add 2% low-molecular-weight agarose solution to a 48-well plate while it is hot, 150 μL per well. After cooling and solidifying at room temperature, inoculate 400 μL of U87 cell suspension per well with a cell density of 2×103 cells/well. Placed in a carbon dioxide incubator, cultured at 37°C, 5% carbon dioxide and saturated humidity for 7 days to form tumor spheres. The BBB/U87 tumor sphere co-culture model was obtained by transferring U87 tumor spheres to the transwell lower chamber of the BBB model;

After LS/FAM, DCDX-LS/FAM, c(RGDyK)-LS/FAM, LCDX/c(RGDyK)-LS/FAM and DCDX/c(RGDyK)-LS/FAM were incubated with 50% serum for 4h, the The liposomes before and after incubation were prepared with DMEM culture solution containing 10% FBS to prepare a solution with a fluorescence concentration of 50 μM. Aspirate the culture solution in the upper chamber of the transwell, add the above solutions respectively, incubate at 37°C for 4 hours, stain the BBB on the transwell with Lysotracker Red for lysosome staining, wash three times with PBS, fix with paraformaldehyde for 15 minutes, stain with DAPI, and place in a co- Colocalization of liposomes and lysosomes was observed under a focusing microscope (Fig. 7). After continuing to culture for 4 hours, the tumor spheres in the chamber were removed and observed under a fluorescent microscope. The results (Fig. 8) show that only the liposomes co-modified by CDX and c(RGDyK) can effectively cross the blood-brain barrier and be taken up by U87 three-dimensional tumorspheres in the lower chamber, and the serum of the DCDX/c(RGDyK)-LS/FAM group The uptake of LCDX/c(RGDyK)-LS/FAM did not change significantly before and after incubation, but LCDX/c(RGDyK)-LS/FAM could not be effectively taken up by tumorspheres in the lower chamber after incubation with serum.

Example 5 In vivo targeting verification of DCDX-LS

Preparation of DCDX/c(RGDyK)-LS/DiR:

The liposome membrane material prescription is the same as above, the above membrane material and DiR are dissolved in chloroform, and the organic solvent is removed by rotary evaporation under reduced pressure to obtain a uniform lipid membrane, which is dried in vacuum for 24 hours. Add physiological saline solution for hydration, and shake in a water bath at 60°C for 2 hours to obtain a liposome suspension. In a water bath at 60°C, use a high-pressure homogenizer (if the liposome volume is less than 10 mL, use a micro extruder) to sequentially squeeze the liposomes through 400, 200, 100 and 50 nm nuclear pore membranes to make the particle size diameter decreases. Then use physiological saline as the eluent to separate and remove unencapsulated DiR through a Sephadex G-50 column to obtain liposomes;

In vivo targeting verification of DCDX/c(RGDyK)-LS/DiR: Orthotopic glioma model mice were injected with LS/DiR, DCDX-LS/DiR, c(RGDyK)-LS/DiR and DCDX/c(RGDyK)-LS/DiR, the dose of DiR was 0.25 mg/kg. After 12 hours, chloral hydrate was anesthetized, the heart was perfused with normal saline, and the fluorescence distribution in the brain was detected with an in vivo imager. The results are shown in FIG. 9 .

Example 6 In vitro pharmacodynamics test of ^D CDX/c(RGDyK)-LS loaded with doxorubicin

The liposome membrane material prescription is the same as above, adopts the ammonium sulfate gradient method to prepare each liposome loaded with doxorubicin (DOX), the dynamic light scattering method measures the particle size distribution, and the liposome morphology is observed by negative staining electron microscopy (as shown in Figure 10 shown); MTT method was used to determine the in vitro effects of free doxorubicin, LS/DOX, ^D CDX-LS/DOX, c(RGDyK)-LS/DOX and ^D CDX/c(RGDyK)-LS/DOX on U87 tumor cells For growth inhibition, U87 cells in the logarithmic growth phase were digested with 0.25% trypsin and blown into single cells. The cells were suspended in DMEM medium containing 10% FBS, counted, and seeded at a density of 3000 cells per well. In a 96-well cell culture plate with a volume of 0.2 mL per well, set aside three wells plus cell-free culture medium as blank wells and culture in a carbon dioxide incubator for 24 hours; use cell culture medium to mix free doxorubicin, LS/DOX, ^D CDX-LS/DOX, c(RGDyK)-LS/DOX and ^D CDX/c(RGDyK)-LS/DOX were serially diluted fourfold. Aspirate the cell culture medium in the 96-well plate, add 200 μL of a series of concentrations of liposome liquid to each well, set up three replicate wells for each concentration, set aside three wells that only add culture medium as control wells, and cultivate for 4 hours Discard the drug solution, wash the plate twice with PBS, add 200 μL of fresh DMEM culture solution to each well, continue to cultivate for 72 h, add 20 μL of MTT reagent (5 mg/mL) to the experimental well, control well and blank well and incubate for 4 h, discard Add 150 μL of dimethyl sulfoxide to each well of the culture solution in the wells, shake to dissolve the blue-purple crystals, measure the absorbance (A) of each well at 490 nm with a microplate reader, and calculate the cell survival rate according to the following formula:

Survival rate=(A _{490 experimental well} -A _{490 blank well} )/(A _{490 control well} -A _{490 blank well} )×100%

Using GraphPad Prism software, the survival rate was plotted against the logarithmic value of the drug concentration (as shown in FIG. 11 ), and the half inhibitory concentration (IC ₅₀ ) was calculated.

Example 7 In vivo pharmacodynamics test of ^D CDX/c(RGDyK)-LS loaded with doxorubicin

Nude mice bearing orthotopic glioma model were injected with normal saline, free doxorubicin, LS/DOX, ^D CDX- LS/DOX, c(RGDyK)-LS/DOX and ^D CDX/c(RGDyK)- LS/DOX. The administration dose was 8 mg/kg, administered on the 6th, 9th, 12th and 15th days after tumor implantation, and the survival time of nude mice was recorded. The survival curve of nude mice is shown in Figure 12. Compared with other groups, ^D CDX/c(RGDyK)-LS/DOX significantly prolongs the survival time of nude mice bearing orthotopic glioma model.

Claims (11)

1. a liposome carrier system for multiple targeted drug delivery modified by a stable polypeptide, characterized in that, it is composed of stable polypeptide ^D CDX and c(RGDyK) and liposomes, and the amino acid sequence of ^D CDX is G ^D R ^D E ^D I ^D R ^D TG ^D R ^D A ^D E ^D R ^D W ^D S ^D E ^D K ^D F. 2. according to the liposome carrier system of the multiple targeted drug delivery of stable polypeptide modification of claim 1, it is characterized in that, its membrane material of described liposome is selected from: a: natural phospholipid or synthetic phospholipid , and b: cholesterol, and c: methoxypolyethylene glycol-phospholipid complex, and d: polypeptide-polyethylene glycol-phospholipid complex containing ^D CDX sequence polypeptide, and e: containing c(RGDyK) sequence Polypeptide-polyethylene glycol-phospholipid complex of polypeptide; the molar ratio of the liposome membrane material is a:b=5:1～1:2, a:c=1000:1～1000:100, a: d=1000:1～1000:100, a:e=1000:1～1000:100. 3. according to the liposome carrier system of the multiple targeted drug delivery of stability polypeptide modification according to claim 2, it is characterized in that, described natural phospholipid or synthetic phospholipid are selected from lecithin, soybean lecithin, hydrogenated soybean lecithin, Dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine, hydrogenated egg phosphatidylcholine, egg yolk sphingomyelin, palmitoyloleoylphosphatidylcholine. 4. The liposome carrier system of multiple targeted drug delivery modified by stable polypeptides according to claim 2, characterized in that in the methoxypolyethylene glycol-phospholipid complex The molecular weight of alcohol is 1000-5000, the molecular weight of polyethylene glycol in the polypeptide-polyethylene glycol-phospholipid complex containing ^D CDX sequence polypeptide is 1000-8000, the polypeptide-polyethylene glycol-phospholipid containing c(RGDyK) sequence polypeptide The molecular weight of polyethylene glycol in the complex is 1000-8000. 5. by the liposome carrier system of the multi-target drug delivery of stable polypeptide modification according to claim 2, it is characterized in that described methoxypolyethylene glycol-phospholipid complex and containing ^D CDX sequence polypeptide The phospholipids in the polypeptide-polyethylene glycol-phospholipid complex and the polypeptide-polyethylene glycol-phospholipid complex containing c(RGDyK) sequence polypeptide are selected from distearoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, hydrogenated Soy Phosphatidylethanolamine, Hydrogenated Egg Phosphatidylethanolamine. 6. according to the liposome carrier system of the multiple targeted drug delivery of stable polypeptide modification according to claim 2, it is characterized in that described polypeptide-polyethylene glycol-containing ^D CDX or c(RGDyK) sequence polypeptide Phospholipid complex, wherein ^D CDX or c(RGDyK) sequence polypeptide is linked with polyethylene glycol-phospholipid to form a complex through covalent bond. 7. The stable polypeptide-modified liposome carrier system for multiple targeted drug delivery according to claim 1, characterized in that the liposome particle size ranges from 50 to 500 nm. 8. Use of the stable polypeptide-modified liposome carrier system for multiple targeted drug delivery according to claim 1 in the preparation of a drug delivery carrier system for the targeted diagnosis and treatment of brain tumors. 9. The use according to claim 8, characterized in that the diagnostic drug contained in the carrier system is selected from the group consisting of fluorescein isothiocyanate FITC, near-infrared dyes Cy5.5, Cy7, IR820, DiR or and magnetic resonance imaging agent Gd. 10. The use according to claim 8, characterized in that the therapeutic drug contained in the carrier system is a small molecule antineoplastic drug selected from the group consisting of adriamycin, epirubicin, paclitaxel, docetaxel, Camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, vincristine, bortezomib, carfenazomib, methotrexate, or cisplatin. 11. The use according to claim 8, characterized in that the therapeutic drug contained in the carrier system is a biological macromolecular anti-tumor drug selected from TRAIL protein, ^L PMI, ^D PMI, TRAIL gene or siRNA.