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Specially-acetylcholine-receptor-targeted D8 polypeptide with biomembrane-crossing effect and brain-targeted drug delivery system thereof

A technology of acetylcholine receptor and drug delivery system, which is applied in the field of pharmacy, can solve the problems of affecting targeting efficiency, hydrolysis, low immunogen and the like, and achieve the effects of good targeting effect and good immunocompatibility.

Active Publication Date: 2020-01-10
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using peptides, proteins or antibodies to interact with their corresponding ligands or antigens to mediate drugs into the brain is currently the main strategy for active targeted drug delivery. Among them, peptides are easy to synthesize, simple in structure, high in affinity and low in immunogen However, natural peptides are composed of L-type amino acids, that is, L-type polypeptides, which are easily hydrolyzed by proteases, which affects the targeting efficiency
According to research in recent years, converting L-type peptides into D-type peptides can make the peptides more stable and improve targeting efficiency, but D-type peptides are difficult to degrade in vivo. , it is easy to cause the body's immune response and generate an immune response. Therefore, how to reduce the immune response caused by the targeting molecule on the premise of ensuring the targeting efficiency is another difficult problem in the research of the targeted drug delivery system.

Method used

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  • Specially-acetylcholine-receptor-targeted D8 polypeptide with biomembrane-crossing effect and brain-targeted drug delivery system thereof
  • Specially-acetylcholine-receptor-targeted D8 polypeptide with biomembrane-crossing effect and brain-targeted drug delivery system thereof
  • Specially-acetylcholine-receptor-targeted D8 polypeptide with biomembrane-crossing effect and brain-targeted drug delivery system thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Preparation of D8, D8-FAM and D8-antitumor drug complex, D8-PEG-DSPE

[0063] Preparation of D8 polypeptide:

[0064] Using solid-phase synthesis, the amino acid sequence was prepared as D RTG D R D A D RE D For the polypeptide (D8) of W, the PAM-amino acid-Boc resin was swelled with DMF for 15 minutes, trifluoroacetic acid (TFA) was deprotected twice, each time for 1 minute, and the Boc-protected amino acid was dissolved in 0.5M HBTU (the solvent was DMF). , react at room temperature for 15 minutes, wash with DMF, remove Boc protection with TFA, and react sequentially according to the amino acid sequence. After the reaction is completed, use 20% piperidine DMF solution to remove the CHO (amino acid sequence containing W) protecting group twice, each time for 15 minutes . After removing the Boc protection by TFA, the polypeptide was cut off from the resin with hydrogen fluoride, and the crude polypeptide was obtained by suction filtration with a sand cor...

Embodiment 2

[0074] Example 2 Stability of D8 and its binding activity test with acetylcholine receptor

[0075] D8 polypeptide stability test:

[0076] Dilute the D8 polypeptide with water to 1mg / mL, mix 0.1mL with 0.9mL 25% sterilized rat serum, and incubate at 37°C at 0.25h, 0.5h, 1h, 2h, 4h, 8h and 12h Take out 100 μL, add 20 μL of acetonitrile containing 0.1% TFA, let it stand at 4°C for 20 minutes, centrifuge at 12000 rpm for 10 minutes, take the supernatant, and measure the amount of D8 polypeptide by RP-HPLC. The experimental results are as follows: figure 1 as shown in a;

[0077] Affinity Determination of D8 Polypeptide and Acetylcholine Receptor:

[0078] Put D8 and D CDX polypeptides were diluted with PBS to different concentrations (10 μM, 25 μM, 50 μM, 100 μM and 200 μM), and after co-incubating with Neuro 2a cells expressing acetylcholine receptors at 4°C for 2 hours, the liposomes loaded with DiI were added D CDX-Lip, with D8 peptide or D The CDX polypeptide competes f...

Embodiment 3

[0079] Example 3 D8 Polypeptide Modified Liposome Trans-Biomembrane Activity Assay

[0080] In vitro verification that liposomes modified with D8 polypeptide can cross the BBB:

[0081] Construction of BBB model in vitro: 4-week-old SD rats were decapitated and the brains were taken out, and the cerebral cortex was quickly separated in ice-cold D-Hank's solution, and the meninges and large blood vessels in the brain were removed, cut into pieces, and collagenase and DNase were added to Digest at 37°C for 90min, centrifuge at 1000r / min for 8min, discard the supernatant, transfer to 20% BSA, centrifuge at 1000g / min4°C for 20min, discard the middle and upper layer liquid, transfer the bottom microvessels to DMEM, and centrifuge at 1000r / min for 5min Finally, the microvascular segment at the bottom was prepared with DMEM culture medium containing 20% ​​fetal bovine serum to prepare a microvascular segment suspension, inoculated on a Transwell plate pre-coated with rat tail collage...

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Abstract

The invention, which belongs to the field of pharmacy, relates to a D8 polypeptide and a brain-targeted drug delivery system thereof, particularly to, a specially-acetylcholine-receptor-targeted D8 polypeptide with a biomembrane-crossing effect and a brain-targeted drug delivery system thereof. According to the invention, a nano-carrier modified by the polypeptide molecule D8 is specifically combined or taken by a positive cell expressing an acetylcholine receptor to realize strong brain targeting and imaging functions and the capability of affinity with a biomembrane barrier to form a cell. With a constructed nano drug delivery system, such as lipidosome, polymer micelle, a polymer disc, and a nanoparticle, an entrapped model drug can be delivered to a brain tissue and a cell expressing an acetylcholine receptor effectively, so that the treatment effect to diseases in brain can be improved obviously; the side effect is low; and the immunocompatibility is high. Therefore, the D8 polypeptide and the brain-targeted drug delivery system thereof have the broad application prospects in fields of diagnosis and targeted therapy of intracerebral diseases such as intracerebral tumors.

Description

technical field [0001] The invention belongs to the field of pharmacy, relates to D8 polypeptide and its brain-targeted drug delivery system, in particular to a targeted polypeptide molecule that can cross biomembrane barriers, especially the blood-brain barrier (BBB), and specifically targets acetylcholine receptors and The D8 polypeptide with transbiofilm effect and its brain-targeted drug delivery system, and its modified complexes and nano drug delivery systems are used in the preparation of preparations for the diagnosis and treatment of peripheral tumors, brain tumors and other brain diseases. The present invention The peptides are more stable in serum, have better targeting effect, and have better immunocompatibility. Background technique [0002] Due to the existence of the blood-brain barrier, it is difficult for traditional drugs to enter the brain, causing a major problem in the clinical treatment of brain diseases. Therefore, active targeted drug delivery systems...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06A61K49/00A61K47/64A61K47/42A61K9/107A61K9/127A61K31/704A61P35/00
CPCC07K7/06A61K49/0056A61K47/64A61K47/42A61K9/1075A61K9/1271A61K31/704A61P35/00
Inventor 占昌友官娟
Owner FUDAN UNIV
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