Intravitreal administration of therapeutics for treating ocular diseases such as age-related macular degeneration and diabetic retinopathy is key in achieving optimal intraocular drug concentration. Diffusion through the hyaluronic acid (HA) component of the vitreous humor governs the transport of an injected protein therapeutic to the target tissues. Liquid rich in HA forms in the vitreous humor at about age 40 and steadily progresses so that by 80-90 years of age, more than 50% of the vitreous is liquid. Hence, the tracking of bovine IgG movement in an in-vitro HA 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 measure diffusion of IgG in an HA matrix formulated to represent the injection site. IgG diffusion in a HA matrix (MW = 0.5 MDa) with a viscosity of 0.1 Pa.s represents the vitreous humor of age groups between 65 and 80; and 1 Pa.s for ages lower than 65. The measurement was done using our previously reported method based on a repurposed cell culture chamber filled with 6 mm deep HA, and in which triplicate injections of 20 µL of label-free bovine IgG at 50 mg/mL were imaged in a gel scanner. Tryptophan residues activated at 280 nm and fluoresced at 384 nm enabled direct measurement of diffusion of IgG in the HA matrix. A significant increase in the diffusion coefficient was observed as the HA matrix viscosity decreased from 1 Pa.s to 0.1 Pa.s. This approach provides a facile method for preclinical screening of candidates for treating ocular diseases as well as for correlating diffusion properties of therapeutics to age-related changes in viscoelasticity of the vitreous humor.
