Phosphorylated protein, intracellular delivery system based on phosphorylated protein, preparation method and application
A technology of phosphorylated protein and delivery system, which is applied in the field of phosphorylated protein, can solve the problems of difficult to design a universal protein delivery system, protein isoelectric point, hydrophilic and hydrophobic properties, etc., to enhance the interaction force , Enhanced electrostatic binding force, high negative charge density effect
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Embodiment 1
[0054] Take RNase A as an example to modify proteins in two steps. The small molecule 4-(hydroxymethyl)phenylboronic acid pinacol carbonyl imidazole (PBA-CDI) was pre-synthesized for use: 4-(hydroxymethyl)phenylboronic acid pinacol ester (7.37 g, 31.5 mmol) was dissolved in Add carbonyldiimidazole (10.2 g, 62.9 mmol) to water DCM (50 mL), and stir overnight at room temperature. Add ethyl acetate (200 mL) to the resulting solution, wash with deionized water (100 mL × 3), and anhydrous MgSO 4 Dry, filter, and remove the solvent by rotary evaporation to obtain the final product PBA-CDI. 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), added PBA-CDI in DMSO solution (79 µL, 10 mg / mL, 2.409 µmol) under stirring, and reacted at room temperature for 20 hours; the reaction ended Afterwards, the reaction solution was transferred to an ultrafiltration tube (MWCO = 10000 Da) and washed with 0.1 M NaHCO 3 (pH = 9.5) solutio...
Embodiment 2
[0057] To study cellular uptake of free proteins and LPP / protein complexes, proteins were labeled with FITC. RNaseA (2 mg, 0.146 µmol, 1.603 µmol -NH 2 ) fully soluble in NaHCO 3 solution (750 µL, 0.1 M, pH = 9.5), followed by a freshly prepared FITC solution in DMSO (250 µL, 4 mg / mL). React at room temperature in the dark for 2 hours. After the reaction is complete, transfer the reaction solution to an ultrafiltration tube (MWCO = 10000 Da) and wash with 0.1 M NaHCO 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, and FITC-RNase A solution is obtained, and stored at -20 °C in the dark , 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 protein R-P-ATP of Example 1 is respectively compounded...
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 xenograft tumor models. When the tumor volume reaches 60 mm 3 , the mice were randomly divided into 4 groups (10 mice in each group), and PBS, free RNase A (1 mg / kg RNase A), LPP / B-P-ATP nanocomplex 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 volume and mouse body weight were measured every other day during the 12-day observation period. see 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 a cell-killing effect. The LPP / R-P-ATP complex can significantly inhibit the growth of tumors during the 12-day observation period, showing the best tumor suppression efficiency. At the same time, the b...
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