Liisa Hirvonen edited Discussion.tex  about 8 years ago

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With anisotropy measurements, the cubic dependence of the radius on the rotational correlation time means that this technique is more sensitive to small changes in radius than methods where the radius depends on the measurement linearly, e.g.\ fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP).  Time-resolved anisotropy measurements can also give information about the shape of the molecule via the model used to fit the decays. The anisotropies measured in this work are clearly double-exponential: besides the component corresponding to the rotation of the drug molecule, there is an additional fast component. However, this fast component cannot be attributed to non-spherical shape of the molecule; in case of an ellipsoid, the anisotropy decay is three-exponential, but the three exponentials are linked, and the fast component is too fast to fit this model.\cite{Lakowicz2006} While it is too fast to be measured accurately with this method due to the long lifetime of the dye, the results indicate a size comparable to the dye molecule, molecule (MW=1~kDa),  and this component is most likely caused by the rotation of the dye molecule on its bond. Wilkins \textit{et al.}\ report a similar component.\cite{Wilkins1999} Double-exponential fitting accounting for this component produces excellent fit results for our experimental data, indicating that a spherical model for the proteins is appropriate for our data analysis. DLS, based on the measurement of fluctuating scattered light intensity due to Brownian motion of the particles, is a well established method for determining the size of small particles in solution, including macromolecular drugs. Wen \textit{et al.}\ \cite{Wen2013} report hydrodynamic radii of 4.2~nm for ranibizumab and 6.3~nm for bevacizumab using DLS, and 5.4~nm for BSA measured as a control, and Li \textit{et al.}\ \cite{Li2011} 4.1~nm, 6.5~nm and 4.8~nm for the same molecules, respectively. These results are slightly higher than estimates based on MW. DLS is limited to very low solute concentrations (generally $\lesssim$1~mg/ml).