Intravitreal administration of therapeutics for treating ocular diseases such as age-related macular degeneration and diabetic retinopathy is key in achieving peak intraocular drug concentration. The transport of protein therapeutics after intravitreal injection to the target tissues is driven by diffusion whereas convection plays a significant role for nanoparticles. Hyaluronic acid (HA) is a major constituent and determines the key attributes of the vitreous humor of the eye. The tracking of bovine IgG movement over time in an in-vitro hyaluronic acid matrix simulates protein diffusion in the vitreous humor and informs a fundamental understanding of the diffusion properties of injected IgG type protein therapeutics.
Our work provides a pre-clinical in-vitro tool to screen protein therapeutics and measure their diffusion in a HA matrix formulated to represent the injection site and correlate it with their in-vivo behavior. Our previously reported work has shown concentration dependent IgG diffusion in a high molecular weight HA matrix (1.5 MDa) that represents the SQ environment. The current work explores IgG diffusion in a low molecular weight HA matrix (0.5 MDa) with a viscosity of 0.2 Pa.s representing the vitreous humor. The measurement was done using our previously reported method where a repurposed cell culture chamber was filled with 6 mm deep HA and the movement of triplicate 20 μL bovine IgG injection in the HA matrix was imaged by using gel imaging scanner. The imaging of bovine IgG is done label-free by activating the tryptophan residues at 280 nm and capturing the protein auto-fluorescence at 384 nm for 3-5 mins. We report the measurement of diffusion coefficient of IgG in a low molecular weight HA matrix by analyzing images of the protein boluses that tracks its movement over time. The IgG diffusion increased from our previously reported values due to the decrease in the molecular weight of the HA matrix.
Both diffusion and convective effects are important for the transport of intravitreal therapeutics. In older population, there is evidence of age-related reduced viscoelasticity of the vitreous due to its liquefaction (synchysis) which influences the in-vivo transport of the injected therapeutic.
Our work provides a facile approach to screening preclinical candidates for ocular diseases as well as correlating diffusion properties of therapeutics to age-related changes in viscoelasticity of the vitreous humor.

Antonio Carlos Freitas dos Santos

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