Multifunctional targeting polypeptide rap and its use in preparing tumor targeting delivery system
A drug delivery system, RAP12 technology, applied in the field of pharmacy, can solve the problems of difficult modification, too large molecular weight, difficult to purify, etc.
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Embodiment 1
[0098] L RAP22 and L RAP12, L RAP22-FAM and L RAP12-FAM, L RAP22-drug complex and L RAP12-drug complex, L RAP22-PEG-PLA and L Synthesis and Characterization of RAP12-PEG-PLA 1) L RAP22 and L Synthesis and characterization of RAP12
[0099] The Boc solid-phase peptide synthesis method was used to synthesize L RAP22 and L RAP12. The specific method is as follows: the amino acids are sequentially connected to the MBHA resin, and the reaction is carried out with HBTU / DIEA as the condensing agent and TFA as the deprotecting agent. After the reaction is completed, the resin is cleaved with hydrogen fluoride containing p-cresol, and the reaction is stirred in an ice bath. After 1 h, the hydrogen fluoride in the tube was removed under reduced pressure after the reaction was completed, and the precipitate was precipitated and washed with glacial ether for 3 times. The crude polypeptide was separated and purified by acetonitrile / water (containing 0.1% TFA) system. Purity an...
Embodiment 2
[0111] Example 2 Polypeptide L RAP22 and L Binding activity of RAP12 and LRP1 protein
[0112] The pre-binding analysis was carried out by the biacore system, and pH 4.5 was selected as the optimal pH for binding the LRP1 protein to the CM5 chip. The recombinant human LRP1 protein was coupled to the CM5 chip, and the RU value reached the target value. L RAP22 and L RAP12 polypeptides were configured into a series of sample solutions with gradient concentrations, injected sequentially from low to high, and analyzed by Biacore T200Evaluation software L RAP22, L The binding activity of RAP12 polypeptide to LRP1 protein, and its binding constant Ka value, dissociation constant Kd value, and affinity constant K were calculated respectively. D value (eg Image 6 shown).
Embodiment 3
[0113] Example 3 Polypeptide L RAP22 and L In vitro cell targeting validation of RAP12
[0114] 1) L RAP22-FAM and L In vitro targeting of RAP12-FAM to glioma cells U87
[0115] Take the monolayer cultured U87 cells in the logarithmic growth phase, digest the monolayer cultured cells with 0.25% trypsin, and prepare a single cell suspension with DMEM medium containing 10% fetal bovine serum at 1 × 10 per well. 5 The cells were seeded in a 12-well culture plate with a volume of 1 mL per well, and the culture plate was transferred to a carbon dioxide incubator at 37°C, 5% CO. 2 After culturing for 24h under the condition of saturated humidity, the concentration of FAM, 5μM was prepared with DMEM medium containing 10% fetal bovine serum. L RAP22-FAM and L RAP12-FAM solution. Aspirate the culture solution in the culture plate, add the above solutions respectively, incubate at 37°C for 4h, and discard the supernatant. Washed three times with PBS solution, fixed cells with fo...
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