Diffusion of nanoparticles in heterogeneous hydrogels as vitreous humour in vitro substitutes

Nanomedicine has the potential to increase the biostability of drugs to treat retinal diseases, improving their performance and decreasing the required number of intravitreal injections. However, accurate pharmacokinetic studies of these nanoparticle-drug conjugates, nanoparticle motion across the vitreous humour and interaction with the retinal cell layers still need to be investigated. Existing nanoparticle tracking techniques require fluorescent labels, which can impact cytotoxicity, nanoparticles' motion, protein interactions, and cell internalization. In this study, a real-time label-free tracking technology, for single nanoparticles in an optical microscope based on the optical phenomena of caustics, was used to characterise the diffusion of nanoparticles in agar-hyaluronic acid hydrogels, previously validated as vitreous humour substitutes for in vitro models. The results demonstrated that the diffusion of nanoparticles through these hydrogels was heterogeneous, and that nanoparticle size had an important role in nanoparticle distribution across and within in vitro vitreous substitutes. These findings suggest that nanoparticle diameter is a critical parameter for designing novel therapeutics for retinal diseases. Moreover, nanoparticle charge did not affect nanoparticle diffusion or distribution in these synthetic hydrogels. The use of caustics in optical microscopy has been demonstrated to be a reproducible, inexpensive technique for screening novel therapeutics in eye in vitro models.