R. Ragheb, E. Esposito, D. Griffiths
Keywords: nanoparticle tracking analysis, liposome, insulin receptors, particle size and concentration
Summary:Aggregation of biological and synthetic materials is a source of tremendous concern, posing problems at various stages of discovery and formulation processes. The ability to identify and characterize aggregates is of broad utility, and experiments described here demonstrate NanoSight’s unique ability to understand dynamics of aggregation in a biological system through high resolution particle-by-particle analysis. For this work, we’ll utilize a model system that has been used in drug discovery and presents some unique challenges in reagent characterization. This system afforded functional assembly of cloned/recombinant membrane receptors on liposomes, without the necessity of reconstitution of full length receptors. Our aim is to correlate previously published activity data with the changes in size and aggregation state of the liposome-RTK complex as measured by Nanoparticle Tracking Analysis (NTA). Liposomes have been used in drug discovery and drug delivery for some time, and the biophysical characterization of these systems and their payloads is critical to understanding their function. One such payload, which we focus on here, are the membrane-associated receptor tyrosine kinase (RTK) targets which continue to be the focus of many discovery campaigns. RTKs are typically single-pass transmembrane signaling proteins which are difficult to purify intact, and are important targets in many disease pathways. The experiments described here represent characterization methods which will be of broad utility to the nanoparticle field. Determination of size, assessment of polydispersity, and identification of components which promote aggregation, are all of interest, and can all be assessed in these simple experiments. The model characterization system was chosen for this work because of the abundance of activity data that exists. For this work, we first investigate the aggregation state of a representative RTK target (kinase domain of insulin receptor). Then we analyze the RTK on an engineered cell-like scaffold, specifically a liposome functionalized with nickel-chelated head groups (described in references 1 and 2). Briefly, nickel head groups facilitate template-directed self-assembly of his-tagged RTKs, such that the kinase domains (cytoplasmic fragments) are oriented as though within the cell. This analysis is performed using magnesium (Mg) or manganese (Mn) as the available counter-ion in the assay buffer, in order to understand why manganese might be increasing the activity level of kinases despite the understanding that manganese is not a physiologically relevant counter-ion. We hope to gain insight into the effects of counter-ion using NTA, and to correlate this to previously obtained activity data. NanoSight from Malvern Instruments presents a unique capability to rapidly characterize this well-studied drug discovery system by directly visualizing nanoscale particles in suspension (between 10 to 2000 nm), with high-resolution, in real-time and with minimal sample preparation. Nanoparticle Tracking Analysis (NTA) software delivers an unprecedented insight into size distributions through a particle-by-particle approach to particle sizing. NanoSight provided visual validation and high resolution analysis of particles within the nanoscale range with both size and concentration measurements. NTA offers a robust analysis of this liposome-protein system and shed light on this method of kinase activation.