Interaction of a Homologous Series of Amphiphiles with P-glycoprotein in a Membrane Environment-Contributions of Polar and Non-Polar Interactions.

The transport of drugs by efflux transporters in biomembranes limits their bioavailability and is a major determinant of drug resistance development by cancer cells and pathogens. A large number of chemically dissimilar drugs are transported, and despite extensive studies, the molecular determinants of substrate specificity are still not well understood. In this work, we explore the role of polar and non-polar interactions on the interaction of a homologous series of fluorescent amphiphiles with the efflux transporter P-glycoprotein. The interaction of the amphiphiles with P-glycoprotein is evaluated through effects on ATPase activity, efficiency in inhibition of [125I]-IAAP binding, and partition to the whole native membranes containing the transporter. The results were complemented with partition to model membranes with a representative lipid composition, and details on the interactions established were obtained from MD simulations. We show that when the total concentration of amphiphile is considered, the binding parameters obtained are apparent and do not reflect the affinity for P-gp. A new formalism is proposed that includes sequestration of the amphiphiles in the lipid bilayer and the possible binding of several molecules in P-gp's substrate-binding pocket. The intrinsic binding affinity thus obtained is essentially independent of amphiphile hydrophobicity, highlighting the importance of polar interactions. An increase in the lipophilicity and amphiphilicity led to a more efficient association with the lipid bilayer, which maintains the non-polar groups of the amphiphiles in the bilayer, while the polar groups interact with P-gp's binding pocket. The presence of several amphiphiles in this orientation is proposed as a mechanism for inhibition of P-pg function.

Exogenous loading of miRNAs into small extracellular vesicles.

Small extracellular vesicles (sEVs), through their natural ability to interact with biological membranes and exploit endogenous processing pathways to convey biological information, are quintessential for the delivery of therapeutically relevant compounds, such as microRNAs (miRNAs) and proteins. Here, we used a fluorescently-labelled miRNA to quantify the efficiency of different methods to modulate the cargo of sEVs. Our results showed that, compared with electroporation, heat shock, permeation by a detergent-based compound (saponin) or cholesterol-modification of the miRNA, Exo-Fect was the most efficient method with > 50% transfection efficiency. Furthermore, qRT-PCR data showed that, compared with native sEVs, Exo-Fect modulation led to a > 1000-fold upregulation of the miRNA of interest. Importantly, this upregulation was observed for sEVs isolated from multiple sources. The modulated sEVs were able to delivery miR-155-5p into a reporter cell line, confirming the successful delivery of the miRNA to the target cell and, more importantly, its functionality. Finally, we showed that the membrane of Exo-Fect-loaded sEVs was altered compared with native sEVs and that enhanced the internalization of Exo-Fect-loaded sEVs within the target cells and decreased the interaction of those modulated sEVs with lysosomes.

Effect of dipole moment on amphiphile solubility and partition into liquid ordered and liquid disordered phases in lipid bilayers.

Association of amphiphiles with biomembranes is important for their availability at specific locations in organisms and cells, being critical for their biological function. A prominent role is usually attributed to the hydrophobic effect, and to electrostatic interactions between charged amphiphiles and lipids. This work explores a closely related and complementary aspect, namely the contribution made by dipole moments to the strength of the interactions established. Two xanthene amphiphiles with opposite relative orientations of their dipole and amphiphilic moments have been selected (Rhodamine-C14 and Carboxyfluorescein-C14). The membranes studied have distinct lipid compositions, representing typical cell membrane pools, ranging from internal membranes to the outer and inner leaflet of the plasma membrane. A comprehensive study is reported, including the affinity of the amphiphiles for the different membranes, the stability of the amphiphiles as monomers and their tendency to form small clusters, as well as their transverse location in the membrane. The orientation of the amphiphile dipole moment, which determines whether its interaction with the membrane dipole potential is repulsive or attractive, is found to exert a large influence on the association of the amphiphile with ordered lipid membranes. These interactions are also responsible for the formation of small clusters or stabilization of amphiphile monomers in the membrane. The results obtained allow understanding the prevalence of protein lipidation at the N-terminal for efficient targeting to the plasma membrane, as well as the tendency of GPI-anchored proteins (usually lipidated at the C-terminal) to form small clusters in the membrane ordered domains.