Experiments on increasing permeability in biological membranes
Wednesday 29 January, 2014
Dr. Brid Cronin, Keble Fellow and Tutor in Chemistry, reports here how her work has benefited from a Keble Small Research Grant.
Electroporation is an approach that is widely used in cell and molecular biology to transiently increase the permeability of the cell membrane by the application of an electric field. The technique is now in clinical use as a method for drug delivery in vivo, and trials are underway to investigate its application as a tumor ablation technique. However, there is relatively little knowledge regarding the mechanism of pore formation on a molecular level. Various models for predicting pore size and shape exist, but the kinetics of pore formation are not well understood. Last summer the ASC supported a Keble student, Jason Sengel, to work with me to understand the thermodynamics of this process and to directly image the pores.
The experiments we have designed are novel and technically demanding. We start by constructing a model cell membrane, achieved by contacting an aqueous droplet of nanolitre volume (Figure 1) with a hydrogel substrate in the presence of an oil and lipid mixture. Lipid molecules assemble on the surfaces of the droplet and the planar hydrogel; gravity serves to bring the two into contact, thus forming a bilayer.
Figure 1. A lipid monolayer is assembled when droplets are prepared in lipid and oil.
An electrode is then inserted into the droplet, and a second into the substrate, allowing the application of varying potentials across the membrane. We monitor the current across the membrane: pore formation results in an increase in the detected current. Simultaneously, we can visualise any pores formed using total internal reflection fluorescence (TIRF) microscopy. A fluorescent dye that emits light in the presence of calcium may be included in the droplet, with the hydrogel on the opposing side of the membrane containing calcium ions. When holes form in the membrane, calcium flows from the substrate into the droplet and we can ‘see’ the pores (Figure 2). The pores are smaller than the diffraction limit, typically between 0.1 and 10 nm.
Figure 2. Electropores in the membrane, visualised by fluorescence due to calcium ions binding to fluo-8H.
The mechanism of pore formation we observe matches well with the simple models proposed in the 1980s. We are now investigating energetic effects of additional cell membrane components on pore formation as well as the affect of membrane fluidity and defects in the lipid membrane structure.
Relevant references on the technique:
JACS, 2011, 133, 14507;
JACS, 2009, 131, 1652.
Dr. Bríd CroninKeble Research Fellow and Tutor in Chemistry.
This article was first published in the ASC Newsletter HT 2012 (March 2012).