Liposomes as potential masking agents in sport doping. Part 2: Detection of liposome‐entrapped haemoglobin by flow cytofluorimetry

Abstract

This work presents an analytical procedure for the identification and characterization of liposome-entrapped haemoglobins, based on flow cytofluorimetry. Flow cytofluorimetric detection is carried out following labelling by two distinct fluorescent reagents, an anti-haemoglobin antibody, fluorescein isothiocyanate conjugated, and an anti-poly(ethylene glycol) antibody, streptavidin-phycoerythrin conjugated. This experimental strategy allows the detection of liposome-entrapped haemoglobins in aqueous media, including plasma; the efficacy of the proposed approach has been verified on whole blood samples added with the liposomal formulation (ex-vivo). Additionally, the proposed technique allows the characterization of several key parameters in the study of liposomal haemoglobins, including, for instance (1) the determination of the degree of haemoglobin entrapment by liposomes; (2) the poly(ethylene glycol) insertion efficiency; and (3) the evaluation of liposome-entrapped haemoglobins stability following storage at 4 °C, allowing to follow both the process of haemoglobin loss from liposomes and the liposome degradation. The procedure is proposed for the detection and characterization of liposome-entrapped haemoglobin formulations to control their misuse in sport, but is also suggested for further applications in biological and clinical laboratory investigations. Copyright © 2016 John Wiley & Sons, Ltd.

Annual banned‐substance review: Analytical approaches in human sports drug testing

“…This analysis yielded a total of 28 compounds that presumably enables a differentiation of the administration of liposome‐containing substances from natural urinary or plasma‐borne lipids as most of them were not present in blank urine and plasma specimens. As the capability of liposomes to act as masking agents is not limited to low molecular mass drugs (eg, anabolic agents), the option to probe for liposome‐encapsulated hemoglobin by flow cytofluorimetry was also assessed . Here, the feature of poly(ethylene glycol) (PEG) insertion into liposomes was exploited enabling targeting of the PEG moiety as well as the hemoglobin content of a particle (the liposome) by means of 2 fluorescent antibodies.…”

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“…Controlled release, understanding target structure and function, achievement of the maximum therapeutic effect, minimized risk to benefit ratios have been the major factors in designing novel drug delivery systems in the past few decades [4]. Nanotechnology has been utilized to treat a variety of disease and disorders and its applications can be traced in physics, chemistry, communication, robotics, biology and medicine [5,6].…”