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\section{Introduction}  There are many diseases that manifest in the posterior segment of the eye. These include age-related macular degeneration (AMD), retinal vein occlusion, and diabetic retinopathy and maculopathy. Together they account for the majority of blind registrations in the developed world.\cite{Bunce2010} Many of these diseases are treated with regular injections of drugs into the vitreous cavity, with the inconvenience of regular clinic review, cost of repeated  injection, discomfort, and small but repeated risks of complications.\cite{Edelhauser2010} Given the many downsides of regular intravitreal injections, the drug industry is actively investigating novel methods of delivering drugs to the posterior segment, including sustained release intravitreal devices,\cite{Callanan2008} transscleral drug delivery,\cite{Ambati2002,Ambati2000a,Ambati2000b} topical drug delivery (eye drops),\cite{Tanito2011} oral \cite{McLaughlin2013} and others such as iontophoresis.\cite{Molokhia2009} Topical drug delivery has many potential advantages, including self-administration, reduced cost, sustained drug levels, potentially fewer clinic visits, and the elimination of the risks associated with eye injections. Whilst desirable, topical drug delivery to the posterior segment is greatly impeded by the external ocular barriers to diffusion. This is compounded by the fact that many of the drugs used to treat posterior segment disease have a high molecular weight (MW), including ranibizumab (Lucentis\textregistered, 48~kDa), aflibercept (Eylea\textregistered, 97~kDa), and bevacizumab (Avastin\textregistered, 150~kDa). 
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R_{min} \text{(nm)} = 0.066 \cdot \text{MW}^{1/3} \label{eq:Erickson}  \end{equation}  However, proteins have a rough surface, are often not perfectly spherical, and their charge affects the diffusion of a molecule in solution. The hydrodynamic radius $R_h$, defined as the radius of a hard sphere that diffuses at the same rate as that solute, takes these effects into account. The hydrodynamic radius is important in predicting transretinal penetration.\cite{Jackson2003,Ambati2000a}   Small-angle scattering studies using X-rays (SAXS) or neutrons (SANS) \cite{Svergun_2013} as well as dynamic light scattering (DLS) \cite{Pecora_1985,Hong_2009} and nuclear magnetic resonance (NMR) techniques \cite{Wilkins1999} have been used for measuring $R_h$. Empirical relationships have been defined between $R_h$ and the number of amino acids $N$ (related to the MW by \(N = \frac{\text{MW}}{110 \text{ Da}}\)), for example, by Wilkins \textit{et al.}\ \cite{Wilkins1999} 
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R_h^D (\text{\AA}) = 1.45\cdot(2.24\cdot N^{0.392}) = 3.248\cdot N^{0.392} \label{eq:Dill}  \end{equation}  These formulas were obtained by global analysis of hundreds of proteins, and fitting to a scatter plot of $R_h$ against MW. However, While these formulas give a good indication of the expected size,  there is a big variance in the measured $R_h$ as a function of MW MW. This can be explained by the diversion of these models from the protein's actual shape, which is  due to molecular shape, charge and surface roughness, and while these formulas give a good indication of the expected size, they do not take these effects into account. roughness.  %\subsection{Radius measurement from anisotropy} 
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\end{equation}  where $\eta$ is the solvent viscosity, $k$ is the Boltzmann constant and $T$ is the absolute temperature.  Although time-resolved anisotropy measurements are a well established tool in molecular biology, only a few studies report applications in ophthalmology.\cite{McLaughlin2013,Danysh2010} In this study the effective hydrodynamic radii of three important posterior segment drugs -- ranibizumab, aflibercept and bevacizumab -- were measured by $\mu$s time-resolved fluorescence anisotropy and compared to radii calculated from the MW. Due to the large molecular weight of these drugs, short-lived nanosecond lifetime fluorescent dyes are not suitable for this study, as the fluorescence will have decayed by the time the drug has moved noticeably. Therefore, we used long-lifetime phosphorescence from a ruthenium based dye \cite{Baggaley2015} which allows us to measure the rotational diffusion of molecules with large molecular weight.\cite{Hoepker2015,Terpetschnig1995} weights.\cite{Hoepker2015,Terpetschnig1995}  A well-characterised protein bovine serum albumin (BSA) of comparable size (66.5~kDa) with known radius \cite{Axelsson1978} (BSA, MW = 66.5~kDa, $R_h$ = 3.48~nm \cite{Axelsson1978})  was also measured to validate the our  results.