Cell-penetrating peptide modified drug carrier as well as preparation method and application thereof
The technology of a membrane-penetrating peptide and a drug is applied in the field of the drug carrier modified by the cell-penetrating peptide and the preparation thereof
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Embodiment 1
[0042] 1. mPEG 5000 -Synthesis and characterization of PLA: take mPEG 5000 (5g) and lactide (4g) were placed in a 50mL dry two-necked bottle, and the water and oxygen in the reaction system were removed by repeated vacuuming and nitrogen filling, and the temperature was slowly raised to 130°C. 5000 After fully melting with lactide, under magnetic stirring, add the toluene solution of 20% (W / V) stannous octoate rapidly as catalyst, wherein the add-on of stannous octoate is 0.2% of reactant gross weight, repeatedly vacuumize to The reaction solution was free of bubbles (to remove toluene, residual moisture and oxygen), and the temperature was raised to 140° C., and reacted for 6 hours under the protection of nitrogen. After the reaction, the two-necked flask was cooled to room temperature, and an appropriate amount of dichloromethane was added to dissolve it. The product was precipitated with ice anhydrous ether under vigorous stirring, and filtered under reduced pressure to ob...
Embodiment 2
[0063] Fluorescent substance coumarin 6 (C 6 ) to simulate drugs, respectively prepare C 6 / PM(5k)-HE-CPP, C 6 / PM(2k)-HE-CPP, C 6 / PM(2k) - CPP and C 6 / PM(2k). MCF-7 cells in the logarithmic growth phase were taken at 2.5×10 5 Each well was inoculated in a 12-well plate, cultured in complete culture solution at 37°C for 48 hours, removed the medium, washed 3 times with PBS (pH 7.4), and added 1 mL of serum-free solution with pH 6.5 and pH 7.4 to each well. medium diluted to C 6 C at a concentration of 100 ng / mL 6 / PM(5k)-HE-CPP, C 6 / PM(2k)-HE-CPP, C 6 / PM(2k) - CPP and C 6 / PM(2k), incubate at 37°C for 2h and 3h, remove the basal culture medium containing the preparation, wash 3 times with PBS at 4°C, add 300μL trypsin to digest, add 600μL complete culture medium to stop the digestion, and gently pipette the cells Transfer to a centrifuge tube, add 600 μL of complete culture solution to wash the 12-well plate, combine after transfer, discard the supernatant after ...
Embodiment 3
[0065] Fluorescent substance coumarin 6 (C 6 ) to simulate drugs, respectively prepare C 6 / PM(5k)-HE-CPP, C 6 / PM(2k)-HE-CPP, C 6 / PM(2k) - CPP and C 6 / PM(2k). Take 4T1 cells in the logarithmic growth phase at 2.5×10 5 Each well was inoculated in a 12-well plate, cultured in complete culture solution at 37°C for 48 hours, removed the medium, washed 3 times with PBS (pH 7.4), and added 1 mL of serum-free solution with pH 6.5 and pH 7.4 to each well. Medium diluted to contain C 6 C at a concentration of 100 ng / mL 6 / PM(5k)-HE-CPP, C 6 / PM(2k)-HE-CPP, C 6 / PM(2k) - CPP and C 6 / PM(2k), incubate at 37°C for 2h and 3h, remove the basal culture medium containing the preparation, wash 3 times with PBS at 4°C, add 300μL trypsin to digest, add 600μL complete culture medium to stop the digestion, and gently pipette the cells Transfer to a centrifuge tube, add 600 μL of complete culture solution to wash the 12-well plate, combine after transfer, discard the supernatant after ...
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