Pgp glo assay

P-gp is an ATP-dependent drug efflux pump that plays an important role in multi-drug resistance and drug bioavailability. The effect of the PEGylated vitamin E isomers on the P-gp ATPase activity was carried out by the Pgp-Glo™ assay (Promega Corporation, Madison, WI). The Pgp-Glo™ assay detects the effects of compounds on recombinant human P-gp in a cell membrane fraction. The assay relies on the ATP-dependent light-generating reaction of firefly luciferase where an increase in luminescence is indicative of the inhibitory effect of compounds on the P-gp ATPase enzyme. PEGylated vitamin E isomers at a final concentration of 10 to 100 μM in assay buffer were added to the wells of a white 96-well plate containing 20 μL (0.5 mM) verapamil, which was required to activate P-gp ATPase (Collnot et al., 2007 (link)). The reaction was initiated by adding Mg ATP (25 mM) to each well. The plate was placed on a shaker for 5 min and then incubated for 40 min at 37 °C. The reaction was stopped by adding 50 μL of the ATP detection reagent. After addition, the plate was left at room temperature for 20 min to allow for the luminescent signal to develop. Luminescence was measured using a Synergy 2 Multi-Mode BioTek plate reader (BioTek Instruments, Inc. Winooski, VT).

The P-gp ATPase activity was evaluated with the P-gp-Glo™ assay (Promega, Co., USA). According to the kit instructions, a series of working solution with P-gp membranes, MgATP and different NanoDDSs at the same Au concentration of 10 μM were prepared in advance and added into the 96-well plate in sequence. Verapamil and sodium orthovanadate were set as positive and negative group, respectively, and all the readings were normalized by the subtraction of negative group.^{22 (link),23 (link)} After incubation at 37 °C for 40 min, the mixture was collected and centrifuged at 12000 rpm for 5 min then rejoined into the 96-well plate. 50 μL of ATP detection reagent was added in each detection well and incubated for an additional 20 min, followed by the luminescence measurement.