A multifunctional peptide mediating paclitaxel delivery and its application
A paclitaxel, multi-functional technology, applied in the biological field, can solve the problems of low encapsulation efficiency, reduced activity, difficult promotion, etc., and achieve the effect of easy quality control and low immunogenicity
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Embodiment 1
[0013] Embodiment 1: the synthesis of multifunctional peptide
[0014] (1) Activated resin: Weigh 2000mg of Fmoc-Tyr wang Resin, add 20mL of DMF and soak for 30min to make it fully swell.
[0015] (2) Deprotection: Press filtration to remove the DMF soaked in the resin, add 10mL of DMF solution containing 20% piperidine, nitrogen blowing and boiling for 15min, then press filtration to remove, remove the Fmoc group of the amino group, use 10mL of isopropanol and Wash the resin three times alternately with 10 mL of DMF, and then use the ninhydrin method to detect that the resin should turn black or purple.
[0016] (3) Condensation reaction: connect the next amino acid, weigh 1.4mmol / g resin Fmoc-amino acid, use 20mL DMF of 910mgTBTU, 0.45g HOBt and 0.52mLDIEA as the reaction liquid, and blow nitrogen at room temperature for 3h, after the reaction , wash the resin three times alternately with 10 mL of isopropanol and 10 mL of DMF. Detect amino groups.
[0017] (4) Steps (2)...
Embodiment 2
[0021] Embodiment 2: Cytotoxicity experiment
[0022] (1) Cell inoculation: take a 96-well plate, add 5×10 3 Cervical cancer Hela cell culture medium, 37 ° C, 5% carbon dioxide incubator for 24 hours, so that the cells adhere to the wall.
[0023] (2) Cultivate cells: Prepare a culture medium containing a multifunctional peptide at a concentration of 1 mM, use a culture medium without a multifunctional peptide as a negative control well (Control), and culture at 37° C. with 5% carbon dioxide for 24 hours.
[0024] (3) Color development: Add 20 μL MTT to each well of 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) Colorimetry: select a wavelength of 490nm, and calculate the cell survival rate by the light absorption value on the microplate reader immunodetector. The results are shown in Table 1.
[0026] Table 1 Multifunctional Peptide Toxicity Test Results
[0027] ...
Embodiment 3
[0029] Example 3: Mediated Paclitaxel Delivery Experiment
[0030] (1) Add culture medium containing 5 μg / mL paclitaxel and 0.1 mM functional 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 culture medium containing only 5 μg / ml paclitaxel was used as a control.
[0031] 2) Add fresh cell culture medium and culture for 24 hours.
[0032] 3) Add 150 μL DMSO to each well of Hela cells, shake for 10 minutes, and then centrifuge at 10,000 rpm to obtain the supernatant to obtain the intracellular extract.
[0033] 4) The concentration of paclitaxel in the intracellular extract was determined by HPLC. The specific test results are shown in Table 2.
[0034] Table 2 Multifunctional peptide-mediated paclitaxel delivery test
[0035]
[0036] It can be seen from Table 2 that the multifunctional peptide of the present invention can efficiently mediate the delivery...
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