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Cell-penetrating peptide with negative charge and application thereof as intracellular transport carrier

A negatively charged, penetrating peptide technology, applied in the field of biomedicine, can solve problems such as low efficiency, and achieve stable metabolism and good intracellular release performance

Inactive Publication Date: 2018-11-02
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its internalization strongly depends on the interaction between positively charged basic amino acids and negatively charged heparan sulfate outside the cell membrane under physiological conditions. At the same time, most of them will be confined to endocytic vesicles after internalization, and the intracellular release process is inefficient

Method used

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  • Cell-penetrating peptide with negative charge and application thereof as intracellular transport carrier
  • Cell-penetrating peptide with negative charge and application thereof as intracellular transport carrier
  • Cell-penetrating peptide with negative charge and application thereof as intracellular transport carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Negatively charged cell penetrating peptide with cellular internalization function and a negative net charge under physiological conditions. The amino acid sequence of the negatively charged cell penetrating peptide is: VELDGDV, LQEDTPP, DPNEKRD, DHMKQHD, NYTWDQW, TTDRHDL, SEQPAHS, DHYHPFS, SDAKNDL, DPYHPFS, SLTNTEH, TVSKGEE, EHYHPFM, TDTTPLQ, GGSAETG, DGPGTAA, SLTAPDY, PLDVNNM, DGTPVGM, LQNLGEF, DSHLSLM, VGSPDGL, DLNLPFA, or PLLEGSL.

[0035] The amino acid sequence VELDGDV has a net charge of -3 under physiological conditions; the amino acid sequence LQEDTPP has a net charge of -2 under physiological conditions; TDTTPLQ, GGSAETG, DGPGTAA, SLTAPDY, PLDVNNM, DGTPVGM, LQNLGEF, DSHLSLM, VGSPDGL, DLNLPFA, and PLLEGSL all had a net charge of −1 under physiological conditions.

[0036] The negatively charged cell-penetrating peptide can internalize mammalian cells, such as human cervical adenocarcinoma Hela cells, human prostate cancer cell line PC-3, human neuroblastoma ce...

Embodiment 2

[0046] In this example, three rounds of whole-cell screening of human cervical adenocarcinoma Hela cells were performed using a phage display peptide library, and 152 cell-penetrating peptide gene sequences were obtained, including 25 negatively charged penetrating peptides. The lower net charge is negative. It has been verified that the above-mentioned negatively charged cell-penetrating peptide can carry enhanced green fluorescent protein and be internalized into the human cervical adenocarcinoma Hela cell line. Its internalization process does not depend on the electrostatic interaction with the cell membrane surface, and is not affected by the pH value. After the cargo is internalized, it forms a diffuse distribution in the cytoplasm, and the intracellular release performance is good, and it does not enter the endosome. Metabolism Stablize.

[0047] The specific process includes the following steps:

[0048] 1) Screening of cell-penetrating peptides by phage display tech...

Embodiment 3

[0072] Use the 25 negatively charged cell-penetrating peptides obtained in Example 2 to verify whether they can internalize the human prostate cancer cell line PC-3, the human neuroblastoma cell line SH-SY-5Y, and the human kidney epithelial cell line Various mammalian cell lines including HEK-293A, mouse embryonic fibroblast cell line 3T3, human umbilical vein endothelial cell line ECV304, and neonatal rat cardiomyocytes. Experimental results show that the 25 negatively charged cell-penetrating peptides of the present invention can internalize the above-mentioned cells.

[0073] Such as figure 1 As shown, the results of experiments showing the internalization of FITC-VELDGDV, FITC-LQEDTPP and FITC-DHYHPFS in different mammalian cells are selected. figure 1 Shown in 5μmol / L FITC-VELDGDV, FITC-LQEDTPP or FITC-DHYHPFS and human prostate cancer cell line PC-3, human neuroblastoma cell line SH-SY-5Y, human kidney epithelial cell line HEK-293A, Fluorescent images of mouse embryon...

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Abstract

The invention provides a cell-penetrating peptide with negative charge and its homologues, having a cell internalization function. Net charge of the cell-penetrating peptide under physiological conditions is a negative value; an amino acid sequence is ELDGDV, LQEDTPP, DPNEKRD, DHMKQHD, NYTWDQW, TTDRHDL, SEQPAHS, DHYHPFS, SDAKNDL, DPYHPFS, SLTNTEH, TVSKGEE, EHYHPFM, TDTTPLQ, GGSAETG, DGPGTAA, SLTAPDY, PLDVNNM, DGTPVGM, LQNLGEF, DSHLSLM, VGSPDGL, DLNLPFA or PLLEGSL. The invention also provides application as an intracellular transport carrier and application of the cell-penetrating peptide withnegative charge as therapeutic drugs in terms of pharmaceutical application and imaging of the intracellular transport carrier.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the sequence characteristics of a class of negatively charged cell-penetrating peptides and their application as a mammalian intracellular delivery carrier in the fields of biology and medicine. technical background [0002] The cell membrane is a semipermeable barrier between the cell and the extracellular environment, with selective permeability so that the cell maintains a constant internal environment. While this phospholipid bilayer is essential for cell survival and function, it poses challenges for the exchange of cargo molecules inside and outside the cell. Since proteins, polypeptides, nucleotides and other pharmaceutical macromolecules and imaging agents must reach the interior of the cell to play their corresponding roles, it is very necessary to realize the transmembrane transport of these substances. [0003] At present, the technologies that can transport macromolecules ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06A61K47/42A61K49/00
CPCA61K47/42A61K49/0056C07K7/06
Inventor 姜勇刘芸罗海华
Owner SOUTHERN MEDICAL UNIVERSITY
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