Imaging of Intra-Matrix IgG Diffusion as an Indicator of Age-Related Vitreous Changes

Intravitreal injection of therapeutics that treat age-related macular degeneration and diabetic retinopathy requires diffusion of the protein therapeutic through hyaluronic acid (HA) in the vitreous humor. The tracking of bovine IgG movement in an in vitro HA matrix simulates protein diffusion in the vitreous humor and provides a fundamental understanding of the diffusion properties of injected IgG type biologics. Phase separation of the vitreous humor, creating pockets of liquid rich in HA, begins at about age 40 and steadily progresses, with more than 50% of the vitreous being liquid by age 80-90 years. When formulated to represent viscosity at the injection site for patients between 65 and 80 years old, the HA has a viscosity of 0.1 Pa s. The vitreous humor for patients at ages lower than 65 corresponds to a viscosity of 1 Pa s. This work shows how HA matrices may be formulated to simulate the HA environment in the vitreous humor so that IgG diffusion may be measured using label-free imaging based on tryptophan autofluorescence. A 37% increase in the mean diffusion coefficient occurred as the HA matrix viscosity decreased from 1 to 0.1 Pa s, thus indicating that protein formulations may need to be adjusted if equivalent diffusion rates are to be achieved in the vitreous humor of different ages of patients. This approach provides an in vitro preclinical screening tool that could prove useful for correlating the diffusion properties of protein therapeutics as a function of the viscoelasticity of the vitreous humor.