Purpose: To measure the hydrodynamic radii of intravitreal anti-VEGF drugs ranibizumab, aflibercept and bevacizumab with \(\mu\)s time-resolved fluorescence anisotropy.
Methods: Ruthenium-based dye Ru(bpy)\(_2\)(mcbpy-O-Su-ester)(PF\(_6\))\(_2\), whose lifetime of several hundred nanoseconds is comparable to the rotational correlation time of these drugs in buffer, was used as a label. The hydrodynamic radii were calculated from the rotational correlation times of the Ru(bpy)\(_2\)(mcbpy-O-Su-ester)(PF\(_6\))\(_2\)-labelled drugs obtained with time-resolved fluorescence anisotropy measurements in buffer/glycerol solutions of varying viscosity.
Results: The measured radii of 2.76\(\pm\)0.04 nm for ranibizumab, 3.70\(\pm\)0.03 nm for aflibercept and 4.58\(\pm\)0.01 nm for bevacizumab agree with calculations based on molecular weight and other experimental measurements.
Conclusions: Time-resolved fluorescence anisotropy is a relatively simple and straightforward method that allows experimental measurement of hydrodynamic radius of individual proteins, and is superior to theoretical calculations which cannot give the required accuracy for a particular protein.
Keywords: Hydrodynamic radius, fluorescence, phosphorescence, time-resolved anisotropy, rotational diffusion