A real‐time in vitro assay to evaluate the release of macromolecules from liposomes

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EarlyView Article

  • Published: Dec 4, 2017
  • Author: Himang Mujoo, John N. J. Reynolds, Ian G. Tucker
  • Journal: Drug Testing and Analysis


The availability of a real‐time assay to experimentally investigate the release of encapsulated proteins would be beneficial given the interest in the use of liposomes as a drug delivery vehicle. Although simple assays for small molecular weight substances exist, assays to evaluate macromolecules do not. Here we describe a method that detects the release of model macromolecules from liposomes in real time. The assay employs the intermolecular distance‐dependent phenomenon of fluorescence resonance energy transfer (FRET) between the fluorophore donor, fluorescein (FITC), and fluorescent quencher, QSY®9. The macromolecular species were conjugated to the markers fluorescein (44kDa dextran) and QSY®9 (67 kDa bovine serum albumin, BSA). Following confirmation of quenching between FITC‐Dex and QSY®9‐BSA, liposomes were loaded with the macromolecular markers and subjected to various treatments (high‐pressure extrusion and Triton X solubilisation) to cause release from liposomes. An increase in FITC fluorescence was observed when liposomes were subjected to extrusion cycles. Surprisingly, the addition of Triton X did not cause an increase in fluorescence probably because the FRET pair became associated with mixed micelles. This assay method should be useful in studies to investigate the mechanisms by which macromolecules are released from liposomes, particularly when liposomes are exposed to release‐triggers (eg, temperature change, pH change, ultrasound). Such understanding will underpin the formulation of triggered liposomal delivery systems for macromolecules.

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