Development of a method to measure protein diffusivities in hyaluronic acid solutions based on a UV-vis spectrophotometer.

Protein diffusion within hyaluronic acid (HA)-rich extracellular matrices can be important for subcutaneous and intravitreal routes of administration. Current techniques for measuring protein diffusivities in HA solutions often require specialized instrumentation and/or complex experimental setups, hindering their widespread application in pharmaceutical laboratories. In this study, by modifying our previous method, we developed a simple, material-sparing approach to determine protein diffusion coefficients in HA solutions using a conventional UV-vis spectrophotometer. By installing a 3D-printed cover with an open slit at the front of a standard UV cuvette, the concentration of a model protein (ovalbumin) at the slit could be measured over time. The effective diffusion coefficient was then calculated from the concentration-time profile using both analytical and numerical methods (the finite difference and finite element methods). As a result, all three methods adequately described the experimental data, with average R² values of approximately 0.99 and comparable diffusion coefficients. Increasing the concentration of high-molecular-weight HA (e.g., 144 kDa and above) reduced ovalbumin diffusivity to a similar degree, independent of HA molecular weights, whereas low-molecular-weight HA caused little inhibition and, under some concentrations, slightly increased protein diffusion. These findings demonstrate that the proposed UV-vis-based method provides an accessible and reliable approach for measuring protein diffusion in HA-containing biological and pharmaceutical matrices.