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Cell-penetrating peptide and application thereof

A technology of membrane-penetrating peptides and functions, which is applied in the application field of membrane-penetrating peptides mediating the delivery of doxorubicin, which can solve the problems of low solubility of doxorubicin, organ toxicity, and low delivery efficiency, so as to facilitate quality control and technical mature effect

Active Publication Date: 2019-04-12
NANYANG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, doxorubicin still has a series of problems such as low solubility, low delivery efficiency and organ toxicity, so the delivery system and means of doxorubicin need to be improved urgently

Method used

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  • Cell-penetrating peptide and application thereof
  • Cell-penetrating peptide and application thereof
  • Cell-penetrating peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0021] Example 2 Cytotoxicity Experiment

[0022] 1) Take a 48-well plate, add 5×10 4 Cervical cancer Hela cell culture medium, 37 ° C, 5% carbon dioxide incubator for 24 hours until the cells adhere to the wall.

[0023] 2) A medium containing a penetrating peptide at a concentration of 1 mM was prepared, and a medium without a penetrating peptide was used as a negative control well (Control), and cultured at 37° C. with 5% carbon dioxide for 48 hours. The medium formula is: DMEM medium containing 10% fetal bovine serum.

[0024] 3) Add 20 μl MTT to each well of the adherent cells, discard the culture medium after continuing to incubate for 4 hours, add 150 μl DMSO to each well, and shake for 10 minutes.

[0025] 4) Select a wavelength of 490nm, and calculate the cell survival rate by the light absorption value on the microplate reader immunodetector.

[0026] Table 1 Penetrating peptide toxicity test

[0027]

[0028] It can be seen from Table 1 that the membrane-pene...

Embodiment 3

[0029] Example 3 Membrane-penetrating peptide and fluorescence detection experiment

[0030] 1) Add a medium containing a certain concentration of FITC and FITC fluorescently labeled penetrating peptide to the adherent cells, incubate for 30min-4h, then carefully suck off the medium, and wash the adherent cells three times with PBS. The medium formula is: 10% DMEM medium.

[0031] 2) Add trypsin to react for 5 minutes until the cells float, and then add fresh medium to stop the reaction.

[0032] 3) Measure the amount of fluorescence possessed by 10,000 cells in a flow cytometer. FITC was used as control and reference (100%).

[0033] Table 2 Multifunctional peptide-mediated doxorubicin delivery test

[0034]

[0035] It can be seen from Table 2 that the total intracellular fluorescence of the FITC fluorescently labeled membrane-penetrating peptide group was geometrically higher than that of the FITC group, and the membrane-penetrating peptide of the present invention pe...

Embodiment 4

[0036] Example 4 Mediated Penetrating Peptide Delivery Experiment

[0037] 1) Add a medium containing 5 mg / ml doxorubicin and 0.1 mM membrane-penetrating peptide to incubate cervical cancer Hela cells. After incubation for 30 minutes, carefully aspirate the medium, and wash the adherent cells three times with PBS. The medium containing only 5 mg / ml doxorubicin was used as a control. The medium formula is: DMEM medium containing 10% fetal bovine serum.

[0038] 2) Add fresh cell culture medium and culture for 24 hours.

[0039] 3) Add 150 μl DMSO to each well of Hela cells, shake for 10 minutes, and centrifuge at 10,000 rpm to obtain the supernatant to obtain the intracellular extract.

[0040] 4) The concentration of doxorubicin in the intracellular extract was determined by HPLC. The results were compared with the doxorubicin control group (100%).

[0041] Table 3 Multifunctional Peptide Mediated Doxorubicin Delivery Test

[0042]

[0043] It can be seen from Table 3 ...

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Abstract

The invention provides a cell-penetrating peptide. The cell-penetrating peptide has an amino acid sequence as follows: Arg-Arg-Lys-Lys-Trp-Trp. The cell-penetrating peptide is capable of mediating delivery of doxorubicin into cells. The cell-penetrating peptide provided by the invention is non-toxic and non-immunogenic. Moreover, the solid-phase synthesis technology of polypeptides is mature, cheap, easy to obtain and conducive to quality control.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a membrane-penetrating peptide and the application of the membrane-penetrating peptide in mediating delivery of doxorubicin. Background technique [0002] Doxorubicin is a broad-spectrum antitumor antibiotic, which can inhibit the synthesis of tumor cell RNA and DNA, and can kill tumor cells in various growth cycles. Because doxorubicin is highly cost-effective, the global sales of doxorubicin reached US$810 million in 2015. However, doxorubicin still has a series of problems such as low solubility, low delivery efficiency and organ toxicity, so the delivery system and means of doxorubicin need to be improved urgently. [0003] Cell-penetrating peptides (CPPs), referred to as penetrating peptides, are a class of short peptides that can efficiently carry macromolecules into cells, and are widely used in the design of new bionanomaterials. Partial membrane-penetrating pept...

Claims

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Application Information

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IPC IPC(8): C07K7/06A61K47/42A61K31/704A61P35/00
CPCA61K47/42A61P35/00A61K31/704C07K7/06
Inventor 韦宇平惠丰立牛秋红宋玉伟
Owner NANYANG NORMAL UNIV
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