Multimodal Investigation into the Interaction of Quinacrine with Microcavity-Supported Lipid Bilayers

Abstract

Quinacrine is a versatile drug that is widely recognized for its antimalarial action through its inhibition of the phospholipase enzyme. It also has antianthelmintic and antiprotozoan activities and is a strong DNA binder that may be used to combat multidrug resistance in cancer. Despite extensive cell-based studies, a detailed understanding of quinacrine’s influence on the cell membrane, including permeability, binding, and rearrangement at the molecular level, is lacking. Herein, we apply microcavity-suspended lipid bilayers (MSLBs) as in vitro models of the cell membrane comprising DOPC, DOPC:Chol(3:1), and DOPC:SM:Chol(2:2:1) to investigate the influence of cholesterol and intrinsic phase heterogeneity induced by mixed-lipid composition on the membrane interactions of quinacrine. Using electrochemical impedance spectroscopy (EIS) and surface-enhanced Raman spectroscopy (SERS) as label-free surface-sensitive techniques, we have studied quinacrine interaction and permeability across the different MSLBs. Our EIS data reveal that the drug is permeable through ternary DOPC:SM:Chol and DOPC-only bilayer compositions. In contrast, the binary cholesterol/DOPC membrane arrested permeation, yet the drug binds or intercalates at this membrane as reflected by an increase in membrane impedance. SERS supported the EIS data, which was utilized to gain structural insights into the drug–membrane interaction. Our SERS data also provides a simple but powerful label-free assessment of drug permeation because a significant SERS enhancement of the drug’s Raman signature was observed only if the drug accessed the plasmonic interior of the pore cavity passing through the membrane. Fluorescent lifetime correlation spectroscopy (FLCS) provides further biophysical insight, revealing that quinacrine binding increases the lipid diffusivity of DOPC and the ternary membrane while remarkably decreasing the lipid diffusivity of the DOPC:Chol membrane. Overall, because of its adaptability to multimodal approaches, the MSLB platform provides rich and detailed insights into drug–membrane interactions, making it a powerful tool for in vitro drug screening.

A cell free biomembrane platform for multimodal study of influenza virus hemagglutinin and for evaluation of entry-inhibitors against hemagglutinin

“…Typical absolute resistance and capacitance of DOPC over MSLB were measured as 2.64 ± 0.2 MΩ and 5.03 µFs m−1 ±0.3 respectively from the EIS studies. The values lie within reported ranges for related biophysical models ( Khan et al, 2013 , 2016 ; Sarangi et al, 2022 ). Here, the observed resistance and capacitance values have not been normalized to the electroactive surface area because of uncertainty in the membrane and electrode area arising from variation in the electrode area or roughness from substrate to substrate.…”

“…Here, the observed resistance and capacitance values have not been normalized to the electroactive surface area because of uncertainty in the membrane and electrode area arising from variation in the electrode area or roughness from substrate to substrate. Such variation is expected during substrate fabrication due to variation in packing in PS sphere over 1 cm × 1.1 cm flat gold electrodes which results in around a 2-5% variation in the electrode area/ roughness as described recently (Sarangi et al, 2022).…”

“…Microcavity arrays ( Scheme 1 ) were prepared on a 1 cm 2 geometric area electrode comprised of one-sided gold deposited on silicon wafer substrate (100 nm gold layer on silicon wafer), ( Maher et al, 2016 ; Basit et al, 2017 ; Gimenez et al, 2020 ; Robinson et al, 2020 ; Sarangi et al, 2022 ), using PS microsphere templating. The SEM image of the gold electrode confirms formation of continuous ordered pore array ( Supplementary Figure S2 ) across the gold substrate.…”

“…The array was then functionalized with SAM of 6-mercaptohexanol at the top surface of the array to help stabilize the lipid monolayer, as reported previously ( Maher et al, 2016 ; Basit et al, 2017 ; Gimenez et al, 2020 ; Robinson et al, 2020 ; Sarangi et al, 2022 ) and described in detail in the earlier section ( Maher et al, 2016 ; Basit et al, 2017 ; Gimenez et al, 2020 ; Robinson et al, 2020 ; Sarangi et al, 2022 ). Thereafter, the pores are buffer filled by sonication and a lipid monolayer assembled by Langmuir Blodgett deposition to form the proximal leaflet followed by vesicle fusion to form the distal monolayer to complete the bilayer.…”

“…We initially compared the relative variation in ΔR after titrating different concentrations of HA with the MSLB containing 1 mol% of GSL mixture (total extract containing GM1, GD1a, GD1b, GT1b) doped at the homogeneous DOPC bilayer with that of a ternary, domain forming composition composed of DOPC:SM: Chol. The absolute resistance and capacitance of ternary composition (DOPC:SM: Chol) forming MSLB before HA incubation were found to be 4.78MΩ ± 0.9 and 4µFs m−1 ±0.4 respectively (Khan et al, 2013(Khan et al, , 2016Sarangi et al, 2022). DOPC:SM:Chol membranes were found to have a greater absolute resistance value and lower capacitance value compared to DOPC membranes, indicating that they were more densely packed.…”