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Acetylcholine receptor-mediated targeting D-configuration polypeptide and application thereof

A technology of acetylcholine receptor and configuration, which is applied to D-configuration polypeptides mediated by acetylcholine receptors and its application fields, can solve the problems of nano-drug delivery system research reports and achieve high binding activity and high stability sexual effect

Active Publication Date: 2015-04-29
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are no D-configuration peptides that can cross the blood-brain barrier or D Research Report on CDX Modified Nano Drug Delivery System

Method used

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  • Acetylcholine receptor-mediated targeting D-configuration polypeptide and application thereof
  • Acetylcholine receptor-mediated targeting D-configuration polypeptide and application thereof
  • Acetylcholine receptor-mediated targeting D-configuration polypeptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] D CDX, D CDX-Fluorescein, D Synthesis and Characterization of CDX-PEG-DSPE

[0068] 1. D CDX and D Synthesis and Characterization of CDX-Cys

[0069] Using the reverse sequence solid-phase peptide synthesis method, designed and synthesized a peptide composed of non-natural D-configuration amino acids D CDX (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 D )and D CDX-Cys (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 D C D ). HPLC and ESI-MS Characterization D CDX and D Purity and molecular weight (Mw) of CDX-Cys. HPLC spectrum, mass spectrogram such as figure 1 , figure 2 shown.

[0070] 2. D Synthesis and Characterization of CDX-Fluorescein

[0071] obtained by the above steps D CDX-Cys was dissolved in 0.1M PBS solution (pH 7.2), Fluorescein-5-maleimide was dissolved in DMF, the two were mixed and reacted with magnetic stirring, monitored by HPLC, and waited for D Af...

Embodiment 2

[0075] D Affinity assay for CDX competitive binding to nicotinic acetylcholine receptors (nAChRs)

[0076] 1. Extraction of rat hippocampal nAChRs membrane protein

[0077] The hippocampus of Wister rats (220-260g) was decapitated and quickly isolated, weighed and added 10 times the volume of Tris-HCl buffer (50mM Tris-HCl, 5mM MgCl 2 ·6H 2 O, 1 mM EDTA, 0.5% (W / V) BSA, 0.1% NaN 3 , 0.32M sucrose, pH7.4), homogenized with a homogenizer at 10,000 rpm, 30 sec each time, 3 times in total. The homogenate was centrifuged at 1000×g for 10 min, and the supernatant was centrifuged at 12,000 rpm at 4°C for 30 min to collect the precipitate, resuspended with 10 times the original weight of Tris-HCl buffer (pH 7.4), and centrifuged for 10 min. Take the precipitate and wash it with the same buffer, centrifuge at 12,000 rpm for 10 min, suspend the precipitate in the above buffer to obtain nAChRs, and store it at -80°C after aliquoting. Protein content was determined by BCA method.

...

Embodiment 3

[0080] Example 3 D CDX Stability Investigation

[0081] 1. D Study on Serum Stability of CDX

[0082] Will D CDX or L CDX was made into a 1mg / mL aqueous solution, 0.1mL was added to 0.9mL of 25% rat serum, incubated at 37°C, 100μL of the reaction solution was taken out at 0.25, 0.5, 1, 2, 4, 8, 12 and 24h, and 20μL of Serum proteins were precipitated with acetonitrile, left at 4°C for 20 minutes, centrifuged at 12,000 rpm for 10 minutes, and 20 μL of the supernatant was taken for HPLC analysis. D Serum stability results for CDX (eg Figure 6 shown) shows that, D CDX has a ratio of L Higher serum stability of CDX.

[0083] 2. D Study on the Stability of Aminopeptidase of CDX

[0084] Will D CDX or L Dissolve CDX polypeptide in 50mM Tris-HCl buffer (pH7.4), incubate with 0.01mg / mL aminopeptidase M at 37°C, sample 100μL at 0, 0.5, 1, 2 and 4h, add 20μL glacial acetic acid After the reaction was terminated, HPLC analysis was performed. D CDX and L Aminopeptidase M s...

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Abstract

The invention belongs to the field of medicine, and relates to D-configuration polypeptide with high stability and capable of realizing mediated targeting of acetylcholine receptor high-expression cells and crossing corresponding barrier membranes and a nano drug delivery system thereof as well as an application in in-vivo and in-vitro brain targeting and in treatment of brain diseases and the like. Test results indicate that DCDX and the acetylcholine receptor are combined with IC50 to obtain 84.5nM which is stable in serum and tolerates hydrolysis of protease; the model drug carried by DCDX is specifically taken in by the positive cells expressing the acetylcholine receptor and has an ability of crossing the barrier formed by the kind of cells; and the nano drug delivery system made of a DCDX-modified polymer carrier material can deliver the entrapped model drug to the target tissue while the drug effect is remarkably improved. The D-configuration polypeptide DCDX provided by the invention can mediate active targeting of the drug or nano drug delivery system and has a good application prospect in the diagnosis and treatment of multiple diseases.

Description

technical field [0001] The invention belongs to the field of pharmacy, and relates to a highly stable D-configuration polypeptide and its nano drug delivery system that can mediate targeting cells with high expression of acetylcholine receptors and cross the corresponding barrier membrane (such as the blood-brain barrier). polypeptide D CDX (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) Nano-drug delivery systems such as liposomes and polymer micelles constructed by their modified polymer carrier materials, as well as in vivo and in vitro brain targeting and applications in the treatment of brain and other diseases. Background technique [0002] Brain disease is a serious disease that endangers human life and health. Due to the existence of the blood-brain barrier, about 98% of small-molecule drugs and almost 100% of large-molecule drugs, including proteins and genes, are difficult to enter the brain, which seriously aff...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08A61K47/42A61K49/00A61K9/127A61K9/107A61K9/00A61K9/14A61P35/00A61P25/28A61K47/34
Inventor 陆伟跃魏晓丽占昌友谢操
Owner FUDAN UNIV
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