Duncan Casey

Drug interactions with lipid membranes

The field of drug-membrane interactions is one that spans a wide range of scientific disciplines, from synthetic chemistry, through biophysics to pharmacology. Cell membranes are complex dynamic systems whose structures can be affected by drug molecules and in turn can affect the pharmacological properties of the drugs being administered. In this tutorial review we aim to provide a guide for those new to the area of drug-membrane interactions and present an introduction to areas of this topic which need to be considered. We address the lipid composition and structure of the cell membrane and comment on the physical forces present in the membrane which may impact on drug interactions. We outline methods by which drugs may cross or bind to this membrane, including the well understood passive and active transport pathways. We present a range of techniques which may be used to study the interactions of drugs with membranes both in vitro and in vivo and discuss the advantages and disadvantages of these techniques and highlight new methods being developed to further this field.

Molecular rheometry: Direct determination of viscosity in Lo and Ld lipid phases via fluorescence lifetime imaging

Understanding of cellular regulatory pathways that involve lipid membranes requires the detailed knowledge of their physical state and structure. However, mapping the viscosity and diffusion in the membranes of complex composition is currently a non-trivial technical challenge. We report fluorescence lifetime spectroscopy and imaging (FLIM) of a meso-substituted BODIPY molecular rotor localised in the leaflet of model membranes of various lipid compositions. We prepare large and giant unilamellar vesicles (LUVs and GUVs) containing phosphatidylcholine (PC) lipids and demonstrate that recording the fluorescence lifetime of the rotor allows us to directly detect the viscosity of the membrane leaflet and to monitor the influence of cholesterol on membrane viscosity in binary and ternary lipid mixtures. In phase-separated 1,2-dioleoyl-sn-glycero-3-phosphocholine-cholesterol-sphingomyelin GUVs we visualise individual liquid ordered (Lo) and liquid disordered (Ld) domains using FLIM and assign specific microscopic viscosities to each domain. Our study showcases the power of FLIM with molecular rotors to image microviscosity of heterogeneous microenvironments in complex biological systems, including membrane-localised lipid rafts.

Targeting of anionic membrane species by lanthanide(III) complexes: towards improved MRI contrast agents for apoptosis

In most healthy mammalian cells an uneven distribution of the mixture of the phospholipid species that make up the bilayer cell membrane is maintained between inner and outer layers: anionic species (principally phosphatidylserine, PS) are arranged largely on the inner layer. 1 In some abnormal cells this is not the case and a considerable amount of anionic lipids are displayed on the outer membrane surface; this is known in cells undergoing the early/intermediate stages of apoptosis (programmed cell death), 2 tumour vasculature, 3 bacteria and viruses. 4 Detection and imaging of apoptotic cells in vivo is desirable, as a clinical and research tool: the extent and speed of onset of apoptosis in tumours following a treatment has shown to be a good prognostic indicator of treatment outcome. 5 In vitro, apoptotic cells are typically detected using biomolecules known to bind phosphatidylserine, conjugated with fluorescent moieties; the most extensively used in this context has been Annexin V. 6 For in vivo imaging, Annexin V and others have been modified with various functionalities for imaging, for

Dynamical hologram generation for high speed optical trapping of smart droplet microtools

This paper demonstrates spatially selective sampling of the plasma membrane by the implementation of time-multiplexed holographic optical tweezers for Smart Droplet Microtools (SDMs). High speed (>1000fps) dynamical hologram generation was computed on the graphics processing unit of a standard display card and controlled by a user friendly LabView interface. Time multiplexed binary holograms were displayed in real time and mirrored to a ferroelectric Spatial Light Modulator. SDMs were manufactured with both liquid cores (as previously described) and solid cores, which confer significant advantages in terms of stability, polydispersity and ease of use. These were coated with a number of detergents, the most successful based upon lipids doped with transfection reagents. In order to validate these, trapped SDMs were maneuvered up to the plasma membrane of giant vesicles containing Nile Red and human biliary epithelial (BE) colon cancer cells with green fluorescent labeled protein (GFP)-labeled CAAX (a motif belonging to the Ras protein). Bright field and fluorescence images showed that successful trapping and manipulation of multiple SDMs in x, y, z was achieved with success rates of 30-50% and that subsequent membrane-SDM interactions led to the uptake of Nile Red or GFP-CAAX into the SDM.

Amphiphilic drug interactions with model cellular membranes are influenced by lipid chain-melting temperature.

Small-molecule amphiphilic species such as many drug molecules frequently exhibit low-to-negligible aqueous solubility, and generally have no identified transport proteins assisting their distribution, yet are able to rapidly penetrate significant distances into patient tissue and even cross the blood–brain barrier. Previous work has identified a mechanism of translocation driven by acid-catalysed lipid hydrolysis of biological membranes, a process which is catalysed by the presence of cationic amphiphilic drug molecules. In this study, the interactions of raclopride, a model amphiphilic drug, were investigated with mixtures of biologically relevant lipids across a range of compositions, revealing the influence of the chain-melting temperature of the lipids upon the rate of acyl hydrolysis.

A Novel, All-Optical Tool for Controllable and Non-Destructive Poration of Cells with Single-Micron Resolution

We demonstrate controllable poration within ≈1 µm regions of individual cells, mediated by a near-IR laser interacting with thin-layer amorphous silicon substrates. This technique will allow new experiments in single-cell biology, particularly in neuroscience.As our understanding of the fundamental mechanistic processes underpinning biology expands, so does the need for high-precision tools to allow the dissection of the heterogeneity and stochastic processes that dominate at the single- and sub-cellular level. Here, we demonstrate a highly controllable and reproducible optical technique for inducing poration within specific regions of a target cell’s plasma membrane, permitting localized delivery of payloads, depolarization and lysis experiments to be conducted in unprecedented detail. Experiments support a novel mechanism for the process, based upon a thermally-induced change triggered by the interactions of a near-IR laser with a biocompatible thin film substrate at powers substantially below that used in standard optoporation experiments.

Optical Tools for Single-Cell Manipulation and Analysis

Experiments on individual cells require a range of extremely precise tools to permit their selection, manipulation, stimulation and analysis. This is further complicated by the cells’ sensitivity to their environment, meaning that such tools must also be very gentle (or at least very localised) to minimise the generation of artefacts. Optical tools provide ideal performance in a number of such roles, exhibiting high spatial and temporal selectivity while causing minimal non-specific effects. This chapter focuses upon the optical tools that have been developed for these purposes, ranging from optical trapping systems which provide a contact-free technique for the manipulation of micron-scale objects, through to a selection of the different optically mediated cell membrane disruption methods available for lysis and/or delivery of material.

The grab-and-drop protocol

We present a rapid and robust technique for the sampling of membrane-associated proteins from the surface of a single, live cell and their subsequent deposition onto a solid-supported lipid bilayer. As a proof of principle, this method has been used to extract green fluorescent protein (EGFP) labelled K-ras proteins located at the inner leaflet of the plasma membrane of colon carcinoma cells and to transfer them to an S-layer supported lipid bilayer system. The technique is non-destructive, meaning that both the cell and proteins are intact after the sampling operation, offering the potential for repeated measurements of the same cell of interest. This system provides the ideal tool for the investigation of cellular heterogeneity, as well as a platform for the investigation of rare cell types such as circulating tumour cells.