title: 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.
authors.affiliation: Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti 1, 00197, Rome, Italy.
authors.forename: Simone
authors.initials: S
authors.lastname: Esposito
authors.affiliation: Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti 1, 00197, Rome, Italy.|Dipartimento di Chimica e Tecnologie del Farmaco, "Sapienza" Università di Roma, Piazzale Aldo Moro 5, 00185, Rome, Italy.
authors.forename: Sonia
authors.initials: S
authors.lastname: Colicchia
authors.affiliation: Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti 1, 00197, Rome, Italy.
authors.forename: Xavier
authors.initials: X
authors.lastname: de la Torre
authors.affiliation: Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti 1, 00197, Rome, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Donati
authors.affiliation: Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti 1, 00197, Rome, Italy.
authors.forename: Monica
authors.initials: M
authors.lastname: Mazzarino
authors.affiliation: Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti 1, 00197, Rome, Italy.|Dipartimento di Medicina Sperimentale, 'Sapienza' Università di Roma, Viale Regina Elena 324, 00161, Rome, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Botrè
mesh_terms: D001802:Blood Substitutes / Q000008:administration & dosage / Q000032:analysis*; D004300:Doping in Sports; D005434:Flow Cytometry; D006454:Hemoglobins / Q000008:administration & dosage / Q000032:analysis*; D006801:Humans; D008081:Liposomes / Q000032:analysis* / Q000097:blood; D010316:Particle Size; D011092:Polyethylene Glycols / Q000032:analysis*; D055550:Protein Stability
chemical_list: D001802:Blood Substitutes; D006454:Hemoglobins; D008081:Liposomes; D011092:Polyethylene Glycols
keywords: blood doping; flow cytofluorimetry; haemoglobin-based oxygen carriers; liposome-entrapped haemoglobin; liposomes; poly(ethylene glycol)