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Targeting delivery system mediated by cholera toxin subunit B protein

A delivery system, cholera toxin technology, applied in preparations for in vivo experiments, medical preparations containing active ingredients, antineoplastic drugs, etc., can solve the problem of restricting the transport of drugs or drug delivery systems and affecting the receptors of pharmacokinetic parameters Combining with ligands, affecting targeted delivery of drugs, etc., to achieve good therapeutic effects

Inactive Publication Date: 2019-01-22
FUDAN UNIV
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Clinical studies have shown that gliomas are different from peripheral tumors. There are different barriers in the occurrence and development of gliomas. For example, in the early stage of tumors, the blood-brain barrier (BBB) ​​remains intact, and common drugs are difficult to treat. Cross the BBB and enter the brain tissue; as the glioma grows, tumor neovascularization forms, and the blood-brain tumor barrier (BBTB) is formed due to the special microenvironment of the brain, while the BBB still exists in the tumor infiltration growth area, Therefore, the delivery of drugs or drug delivery systems to tumor tissues is limited
[0003] Researchers in this field believe that the active targeted drug delivery system is crucial in the treatment of glioma; using peptides, proteins or antibodies to interact with their corresponding ligands or antigens to mediate drugs into the brain is the current active targeted drug delivery system. The main strategy of drug delivery, however, through a single ligand, mediating drugs across the blood-brain barrier and simultaneously targeting new blood vessels and tumor cells still faces great challenges, and the targeted drug delivery system has multiple obstacles in the blood circulation process in vivo, For example, proteins and enzymes in the blood, studies have shown that after the drug delivery system enters the blood circulation, a large number of plasma proteins interact with the nano drug delivery system to form a protein corona, which can affect its pharmacokinetic parameters, receptors and ligands. Combined, even directly taken up by the monocyte-macrophage system, ultimately affecting the targeted delivery of drugs

Method used

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  • Targeting delivery system mediated by cholera toxin subunit B protein
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  • Targeting delivery system mediated by cholera toxin subunit B protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1 CTB protein crosses BBB experiment

[0061] In vivo experiments confirmed that fluorescein FITC-labeled CTB protein (CTB-FITC) can cross the blood-brain barrier:

[0062] Fluorescence-labeled CTB-FITC (2mg / kg) was injected into the tail vein of C57BL / 6 mice. After 1h, the mice were anesthetized with ether, and the brain tissue was taken out, fixed in 4% paraformaldehyde for 24h, 30% Sucrose dehydration, OCT embedding, frozen section, DAPI stained cell nuclei, observed under a microscope and photographed. At the same time, BSA, which was close to the molecular weight of CTB protein, was used as a control and processed according to the same steps. The experimental results showed that the CTB-FITC group was significantly higher than BSA-FITC group, indicating that CTB protein can cross the blood-brain barrier into the brain (such as figure 1 shown);

[0063] In Vitro Validation I 125 Labeled CTB protein (CTB-I 125 ) can cross the blood-brain barrier:

[0...

Embodiment 2

[0067] Example 2 Preparation and Characterization of Nanoparticles

[0068] Preparation of nanoparticles NP / DiI and CTB-NP / DiI:

[0069] Preparation of nanoparticles CTB-NP / DiI: Weigh the film material PLGA: 16mg; mPEG-PLGA: 4mg; mPEG-DSPE: 4mg; DiI: 0.3mg, dissolve in 0.5ml CH 2 Cl 2 Add 2ml of 0.5% sodium cholate solution, sonicate, emulsify (300w, sonicate for 5s, stop for 1s, 60 times in total), vacuum suspension steam, remove organic solvent, 3000rpm, centrifuge for 10min to remove larger precipitates, 12000rpm, centrifuge for 25min, Discard the supernatant, dissolve the nanoparticles in 1ml double distilled water to obtain NP / DiI;

[0070] Preparation of nanoparticles CTB-NP / DiI: Weigh the film material PLGA: 16mg; mPEG-PLGA: 4mg; mPEG-DSPE: 4mg; Mal-PEG-DSPE: 0.2mg; DiI: 0.3mg, dissolve in 0.5ml CH 2 Cl2 Add 2ml of 0.5% sodium cholate solution, ultrasonication, emulsification (300w, ultrasonication for 5s, stop for 1s, 60 times), suspension steaming, removal of orga...

Embodiment 3

[0085] Effects of nanoparticles on CTB protein activity and its interaction with monocyte-macrophage system after protein corona formation in serum

[0086] Binding activity of CTB-NP to GM1 receptor before and after serum incubation:

[0087] The GM1-ELISA kit can quantify CTB protein and detect its activity. The specific operation is as follows: add 2 μg GM1 protein to each well of the ELISA plate, overnight at room temperature, wash with PBS for 3 times, block with 1% BSA for 1 hour, and absorb the BSA solution, add the nanoparticles that were pre-incubated with serum and serially diluted, incubate at 37°C for 1h, wash with PBS three times, add anti-CTB antibody and react at 37°C for 1h, wash with PBS three times, add the corresponding horseradish peroxide Enzyme-labeled secondary antibody, react with TMB chromogenic solution for 3-15min after 1h, and use 2M H 2 SO4 terminated the reaction and measured its absorbance value at a wavelength of 450nm. The experimental results...

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Abstract

The invention belongs to the field of pharmacy, and relates to a targeting delivery system mediated by cholera toxin subunit B protein. In the system, cholera toxin subunit B protein CTB is used for modifying a fluorescence probe, pharmaceutical molecules and a nanometer delivery system through covalent bonds, and the experiment result shows that medicines carried by the CTB protein are specifically taken by GM1-expressing tumor cells, new vascular endothelial cells and brain capillary endothelial cells forming a blood-brain barrier; the nanometer delivery system modified by the CTB protein can effectively deliver the medicines to the focus part, so that the treatment effects of the medicines can be obviously improved; and the CTB protein has high stability in the plasma, and after the delivery system modified by the CTB protein forms protein corona after acting with plasma protein, the take-in and removal of mononuclear macrophages are not increased. The CTB protein can simultaneouslymediate the medicines to span the blood brain barrier and target to the tumor new vessels and tumor cells, and meanwhile, the medicines and the delivery system modified by the CTB protein can be usedfor diagnosing and treating peripheral tumor, brain tumor and other brain diseases.

Description

technical field [0001] The invention belongs to the field of pharmacy, and relates to a targeted drug delivery system, in particular to a targeted delivery system mediated by cholera toxin B chain protein, in particular to a highly stable drug that can simultaneously target tumor neovascularization and tumor cells and A multifunctional protein-modified complex and a targeted delivery system capable of mediating drugs crossing the blood-brain barrier, the targeted delivery system can be used in the diagnosis and treatment of peripheral tumors, brain tumors and other brain diseases. Background technique [0002] The prior art discloses that glioma is one of the most common malignant tumors in the central nervous system. The traditional treatment method is mainly surgical resection, but due to the complexity of the functional area of ​​the central nervous system and the invasive growth of tumor cells, surgery is difficult to completely eradicate tumor tissue. At present, chemo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K49/00A61K45/00A61P35/00
CPCA61K45/00A61K49/0056
Inventor 占昌友官娟
Owner FUDAN UNIV
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