Methylene blue (MB) is FDA-approved and the interaction with the important anticoagulant heparin has been investigated spectroscopically in water. However, details of the interaction in physiological conditions remain unclear. Here, we provide a mechanistic investigation to study the binding kinetics between MB and heparin in phosphate buffer saline (PBS). We noted a 0.04 ppm chemical shift of the proton in the phenothiazine ring of MB in the nuclear magnetic resonance spectrum suggesting electron delocalization and self-aggregation of MB. The absorbance peak of 0.15 mM MB blue shift from 680 to 610 nm upon addition of heparin, even at a high heparin concentration (i.e. 10 U/mL) (Panel A). This indicated that MB formed dimers rather than higher order aggregates as in water. This is because sodium chloride causes dissociation. In addition to MB self-aggregation, we also observed nanoscale MB-heparin hybrid aggregates in transmission electron microscope (TEM) (Panel B). The dynamic light scattering (DLS) analysis showed that the aggregate size (100 nm-2 µm) was governed by MB concentration rather than the heparin (Panel C). We attributed the formation of aggregates to the bridging effect of MB that connected two heparin chains sufficiently bound with MB (Panel D). To verify our hypothesis, we used molecular dynamics simulations to analyze the binding energy, the energy decomposition, and the MB concentration dependent binding activity of MB and heparin.

Mechanistic investigation of methylene blue and heparin interaction in phosphate buffer saline

256th National Meeting (2018)

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