Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Cell-penetrating peptide and preparation method and application thereof

A technology for solid-phase synthesis of membrane-penetrating peptides and polypeptides, applied in the biological field, can solve the problems of erythromycin being difficult to penetrate, and achieve the effects of easy production and promotion, mature technology, and expanded application range

Active Publication Date: 2019-05-10
NANYANG NORMAL UNIV
View PDF3 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The invention provides a membrane-penetrating peptide to solve the problem that erythromycin is difficult to penetrate the blood-brain barrier in the prior art

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Cell-penetrating peptide and preparation method and application thereof
  • Cell-penetrating peptide and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: Synthesis of Penetrating Peptides

[0018] 1) Activation of the resin: Weigh 1500mg of Fmoc-Pro wang Resin, add 25mL of DMF and soak for 10min to make it fully swell.

[0019] 2) Deprotection: Remove the DMF soaking the resin by suction filtration, add 25mL DMF solution containing 20% ​​piperidine, blow and boil with nitrogen for 25min, then remove by suction filtration, wash the resin three times alternately with 25mL isopropanol and DMF, and then use ninhydrin The resin should be black or purple when detected by the method.

[0020] 3) Condensation reaction: connect the next amino acid, weigh 1.4mmol / g resin Fmoc-amino acid, use 910mgTBTU, 0.45g HOBt and 0.52ml DIEA in 25mL DMF as the reaction solution, and react with nitrogen blowing at room temperature for 3h. After the reaction was completed, the resin was alternately washed three times with 25 mL of isopropanol and DMF. Detect amino groups.

[0021] 4) Repeat steps 2)-3) process: extend the polypepti...

Embodiment 2

[0024] Embodiment 2: Cytotoxicity experiment

[0025] 1) Take a 96-well plate and add 7×10 3 Cervical cancer Hela cell culture medium, 37 ° C, 5% carbon dioxide incubator for 36 hours, so that the cells adhere to the wall.

[0026] 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.

[0027] 3) Add 20 μl MTT to each well of adherent cells, discard the culture medium after continuing to incubate for 2 hours, add 150 μl DMSO to each well, and shake for 15 minutes.

[0028] 4) Select a wavelength of 490nm, and calculate the cell survival rate by the light absorption value on the microplate reader immunodetector.

[0029] Table 1. Penetrating peptide toxicity test

[0030]

[0031] It can be seen from Table 1 that the membrane-penetrating peptide of the present invention has no obvious damage t...

Embodiment 3

[0032] Implementation Example 3: Mediated erythromycin delivery experiment

[0033] 1) Preparation of erythromycin complex: adding 5 μg / ml erythromycin and 0.1 mM penetrating peptide in physiological saline and co-incubating for 30 min;

[0034] 2) verify the performance of the resulting erythromycin complex:

[0035] Experimental group: Inject 0.1 ml of the erythromycin complex obtained in step 1) into the mouse tail vein

[0036] Control group: Inject 0.1 ml of normal saline containing only 5 μg / ml erythromycin into the tail vein of mice.

[0037] 3) After 5 minutes of injection, the mice were killed to take brain tissue, the brain tissue was frozen and homogenized, and the erythromycin in the brain tissue was extracted with methanol.

[0038] 4) The concentration of erythromycin in the brain tissue extract was determined by HPLC. Take the content of the control group as a reference (100%)

[0039] Table 2. Penetrating peptide-mediated blood-brain barrier test

[0040] ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to the technical field of biology, in particular to a cell-penetrating peptide and a preparation method and application thereof. The amino acid sequence of the cell-penetrating peptide is Arg-Arg-Leu-Ser-Tyr-Thr-Lys-Lys-Lys-Trp-Trp-Pro. The cell-penetrating peptide is free of physiological toxicity and can be combined with erythrocin under the non-covalence effect, and the erythrocin is effectively mediated for penetrating a blood brain barrier.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a membrane-penetrating peptide and its preparation method and application. Background technique [0002] Erythromycin is a macrolide antibiotic, which is effective against Gram-positive bacteria: Staphylococcus, Streptococcus pyogenes, Streptococcus viridans, Streptococcus pneumoniae, Streptococcus faecalis, Streptococcus hemolyticus, Clostridium difficile, Bacillus diphtheriae, Bacillus anthracis has a strong inhibitory effect. The mechanism of action of erythromycin is to combine with the 50S subunit of ribonucleosomes of pathogenic bacteria, thereby inhibiting peptidyltransferase, and then affecting the translocation process of ribonucleosomes, inhibiting the synthesis of bacterial proteins, and achieving bacterial inhibition. Although erythromycin is the current first-line antibiotic, it is difficult to pass through the blood-brain barrier, which seriously affects its applicatio...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K1/04C07K1/113A61K47/42A61K31/7048A61P25/00
Inventor 韦宇平惠丰立牛秋红鲁云风
Owner NANYANG NORMAL UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products