Construction of P-glycoprotein incorporated tethered lipid bilayer membranes

Abstract

To investigate drug-membrane protein interactions, an artificial tethered lipid bilayer system was constructed for the functional integration of membrane proteins with large extra-membrane domains such as multi-drug resistance protein 1 (MDR1). In this study, a modified lipid (i.e., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000] (DSPE-PEG)) was utilized as a spacer molecule to elevate lipid membrane from the sensor surface and generate a reservoir underneath. Concentration of DSPE-PEG molecule significantly affected the liposome binding/spreading and lipid bilayer formation, and 0.03 mg/mL of DSPE-PEG provided optimum conditions for membrane protein integration. Further, the incorporation of MDR1 increased the local rigidity on the platform. Antibody binding studies showed the functional integration of MDR1 protein into lipid bilayer platform. The platform allowed to follow MDR!-statin-based drug interactions in vitro. Each binding event and lipid bilayer formation was monitored in real-time using Surface Plasmon Resonance and Quartz Crystal Microbalance-Dissipation systems, and Atomic Force Microscopy was used for visualization experiments.

Keywords: Drug–protein interactions; P-glycoprotein; Statins; Tethered lipid bilayer.

Graphical abstract

Fig. 1

Construction of tBLM on gold-coated surface. (A) Activation of the surface with DTSP. (B) DSPE-PEG modification. (C) Construction of protein-free tBLM. (D) Construction of protein-incorporated tBLM (molecules were not presented in their actual sizes).

Fig. 2

Binding and spreading of MDR1-incorporated liposomes at various MDR1 volumes on 0.03 mg/mL of DSPE-PEG-modified surfaces by (A) SPR (● 0.7; ∎ 0.9; ♦ 1.0; ▲ 2.0;▼ 3.0; ✶ 4.0; ◄ 5.0 µL of MDR1) and QCM–D (● 0.7; ∎ 0.8; ♦ 0.9; ▲ 1.0;▼ 2.0; ✶ 3.0; ◄ 4.0 µL of MDR1) (overtone number: 7), (B) using frequency and (C) dissipation parameters.

Fig. 3

Visualization of DSPE-PEG modified surface and liposome binding/spreading using AFM. Topography images of DSPE-PEG layer with (A) 3 nm and (B) 6 nm of vertical scale. Topography image of (C) MDR1-free tBLM and (D) MDR1-integrated tBLM. Dissipation image of (E) MDR1-integrated tBLM.

Fig. 4

Antibody and pravastatin binding on MDR1-incorporated tBLM by SPR. (A) (●) Binding result of anti-MDR1 monoclonal antibody on MDR1-incorporated tBLM. (■) Binding result of anti-MDR1 antibody on MDR1-free tBLM. (▲) Binding result of anti-Pin-1 (G8) mouse monoclonal antibody on MDR1-incorporated tBLM. (B) Binding result of (○) 0.05 mg/mL and (□) 0.01 mg/mL pravastatin solutions on MDR1-incorporated tBLM and binding of 0.05 mg/mL of pravastatin (∆) on MDR1-free bilayers.