Molecular interactions between charged macromolecules: colorimetric detection and quantification of heparin with a polydiacetylene liposome

Abstract

The molecular interactions between an artificial liposome decorated with cationic sites and heparin, a biomacromolecule with dense negative charge, have been studied for the first time. Heparin is used as an anticoagulant during cardiovascular surgery or to avoid thrombosis. Overdose of heparin, however, can induce complications such as hemorrhage or thrombocytopenia, making it important to monitor the amount of heparin in an easy and simple manner. We report a colorimetric assay method for heparin, based on the stimuli-sensitive polydiacetylene liposome. The electrostatic interactions between heparin and the liposome led to a ratiometric colour change from blue to red. Sigmoid curves were obtained from the UV-Vis titrations, from which linear calibration curves were extracted. Addition of anionic biopolymers such as chondroitin 4-sulfate or hyaluronic acid to the same liposome system led to small colour changes. The sensing protocol was successfully applied to the determination of heparin in a HEPES buffer and in a buffer containing fetal bovine serum, for the concentration ranges of 0.30–5.35 U mL⁻¹ and 0.67–4.33 U mL⁻¹ respectively, which covers most part of therapeutic level. The macromolecular ionic interactions proceeded through a sequential morphological change, showing precipitation around the endpoint of the titration and then dissolution of the precipitates later, which was analyzed by dynamic light scattering and transmission electron microscopy. The aggregation behaviour was supported by zeta potential changes during the titration. The zeta potential change occurs at the colorimetric endpoint during the titration of the liposome with heparin, from which a new and simple heparin quantification method is developed.