Novel cell-penetrating peptide mediating drug delivery and application of cell-penetrating peptide
A drug and anti-tumor drug technology, which is applied to the field of membrane-penetrating peptides and the application of the membrane-penetrating peptides in drug delivery and tumor treatment, can solve the problems of low introduction efficiency, difficulty in promotion, death, etc., and achieve efficient mediated delivery, best therapeutic effect
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Embodiment 1
[0042] Example 1: Synthesis of penetrating peptide
[0043] 1) Activated resin: Weigh 1000mg wang resin, add 10-15ml (immersed all resin) DMF and soak for 30min to make it fully swell;
[0044] 2) Deprotection: the DMF soaked in the resin is removed by pressure filtration, 10ml of DMF solution containing 20% piperidine is added, the reaction is boiled with nitrogen for 15 minutes, and then filtered off by pressure to remove the FMOC group of amino groups, and the resin is washed with 10ml of isopropanol Three times, then wash with 10ml DMF three times, and then use the ninhydrin method to detect that the resin should be black or purple;
[0045] 3) Condensation reaction: connect the next amino acid, weigh the amount of Fmoc-amino acid 1.4mmol / g resin, 910mg TBTU, add 10ml DMF and 0.45g HOBt and mix well, add 0.52ml DIEA to make the reaction solution, and nitrogen at room temperature Blowing reaction for 2h. After the reaction, the resin was washed three times with isopropanol, an...
Embodiment 2
[0050] Example 2: Cytotoxicity test
[0051] 1) Take a 96-well plate and add 7×10 to each well 3 Cells, 37℃, 5% carbon dioxide incubator for 24 hours, make the cells adhere to the wall;
[0052] 2) Prepare a culture medium containing different concentrations of SEQ ID No. 3 penetrating peptide, use the culture medium without penetrating peptide as a negative control well (Control), incubate at 37° C., 5% carbon dioxide for 1-5 h;
[0053] 3) Add 20μl MTT to each well of adherent cells, then discard the culture medium after incubating for 24h, add 150μl DMSO (dimethyl sulfoxide) to each well and shake for 10min;
[0054] 4) Choose the wavelength of 490nm or 570nm, and calculate the cell survival rate by light absorption value on the microplate reader immunodetector. The results are shown in Table 1. The results show that the multiple penetrating peptides of the invention have no obvious cytotoxicity. The result of selecting SEQ ID No: 3 is plotted as figure 1 .
[0055] Table 1. The e...
Embodiment 3
[0057] Application Example 3: Penetrating peptide penetration and fluorescence detection experiment
[0058] 1) Add a medium containing 100μM FITC fluorescently labeled SEQ ID No. 2 penetrating peptide to the adherent cervical cancer (Hela) cells, incubate for 30min-4h, carefully aspirate the medium, and wash the adherent cells three times with PBS ;
[0059] 2) Add fixative, 10min-20min, fix the cells and wash the cells 2-3 times with PBS;
[0060] 3) Add DAPI staining solution to stain for 5min-10min and wash 2-3 times with PBS;
[0061] 4) Observe with a fluorescence microscope under the excitation light of 488nm and 543nm respectively, the results are as follows figure 2 . The results showed that the penetrating peptide successfully penetrated the membrane and delivered the fluorescent marker into the cell.
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