Phosphorylated protein, intracellular delivery system based on phosphorylated protein, preparation method and application
A phosphorylated protein and delivery system technology, applied in the field of phosphorylated proteins, can solve the problems of large differences in protein isoelectric point, hydrophilicity and hydrophobicity, and it is difficult to design a universal protein delivery system, so as to enhance the interaction force , electrostatic binding force enhancement, the effect of restoring activity
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Embodiment 1
[0054] Take RNase A as an example to modify proteins in two steps. Pre-synthesized small molecule 4-(hydroxymethyl)phenylboronic acid pinacol carbonylimidazole (PBA-CDI) for later use: 4-(hydroxymethyl)phenylboronic acid pinacol ester (7.37 g, 31.5 mmol) was dissolved in In aqueous DCM (50 mL), carbonyldiimidazole (10.2 g, 62.9 mmol) was added, and the mixture was stirred at room temperature overnight. To the resulting solution was added ethyl acetate (200 mL), washed with deionized water (100 mL × 3), anhydrous MgSO 4 After drying, filtering, and rotary evaporation to remove the solvent, the final product PBA-CDI was obtained. First RNase A (2 mg, 0.146 µmol, 1.603 µmol -NH 2 ) was dissolved in phosphate buffer (PB, 921 µL, 10 mM, pH = 7.4), PBA-CDI DMSO solution (79 µL, 10 mg / mL, 2.409 µmol) was added under stirring, and the reaction was performed at room temperature for 20 hours; the reaction was over After that, the reaction solution was transferred to an ultrafiltratio...
Embodiment 2
[0057] To study cellular uptake of free protein and LPP / protein complexes, proteins were labeled with FITC. First RNase A (2 mg, 0.146 µmol, 1.603 µmol -NH 2 ) is fully soluble in NaHCO 3 solution (750 µL, 0.1 M, pH = 9.5), followed by the addition of freshly prepared FITC in DMSO (250 µL, 4 mg / mL). The reaction was carried out at room temperature for 2 hours in the dark. After the reaction, the reaction solution was transferred to an ultrafiltration tube (MWCO = 10000 Da), and 0.1 M NaHCO was used for the reaction. 3 (pH = 9.5) solution to wash off excess FITC (4000 × g, 4 °C, 10 min, 5 times in total) until the solution outside the ultrafiltration tube is colorless to obtain FITC-RNase A solution, which is stored at -20 °C in the dark , used for subsequent reactions; FITC-R-P-ATP was prepared according to the method of Example 1.
[0058] Cyt C and BSA were fluorescently labeled by the same method to obtain FITC-C and FITC-B.
[0059] The water-soluble phosphorylated pro...
Embodiment 3
[0067] will contain 5 x 10 5 B16F10 cells in PBS (50 µL) were subcutaneously injected into the right back of C57BL / 6 mice to establish a xenograft model. When the tumor volume reaches 60 mm 3 When the mice were randomly divided into 4 groups (10 mice in each group), PBS, free RNase A (1 mg / kg RNase A), and LPP / B-P-ATP nanocomposite were injected intratumorally on the 1st, 3rd, and 5th day, respectively. (1 mg / kg BSA), LPP / R-P-ATP nanocomplex (1 mg / kg RNase A). Tumor volumes and mouse body weights were measured every other day during the 12-day observation period. see the results Figure 7 . Free RNase A did not have any anti-tumor activity, and the tumor growth was similar to that of the PBS group, indicating that free RNase A was difficult to be taken up by tumor cells and exert cell killing effect. The LPP / R-P-ATP complex could significantly inhibit tumor growth during the 12-day observation period, showing the best tumor inhibition efficiency. At the same time, the bo...
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