Influence of anti-glaucoma drugs on uptake of extracellular vesicles by trabecular meshwork cells

Background: Extracellular vesicles (EVs) are capable of manipulating cellular functions for the maintenance of biological homeostasis and disease progression, such as in glaucoma disease. These nano-particles carry a net negative surface charge under physiological conditions that can contribute to EVs:EVs interaction and their uptake by target cells. Purpose: To investigate the effect of glaucoma drugs on EVs physicochemical characters and the implications for their uptake by trabecular meshwork (TM) cells. Methods: TM or non-pigmented ciliary epithelium (NPCE) cells derived EVs were incu-bated with commercial anti-glaucoma formulation, Timolol maleate, Brinzolamide or Benzalkonium Cl and their size and zeta potential (ZP) and physical interactions of EVs derived from NPCE cells and TM cells were evaluated. The contribution of EVs interactions to up-take by TM cells was examined using fluorescence-activated cell sorting. Results: EVs size and ZP were affected by the ionic strength of the buffer rather than EVs type. Commercial glaucoma eye drops, including β-blocker, α-2-agonist and prostaglandin analogs, reduced NPCE EVs ZP, whereas exposure of EVs to carbonic anhydrase inhibitor caused an increase in the ZP. A correlation was found between increased ZP values and increased NPCE EVs uptake by TM cells. We were able to show that Benzalkonium chloride stands behind this ZP effect and not Timolol or Brinzolamide. Conclusion: Altogether, our findings demonstrate that EVs size, surface membrane charge, and ionic strength of the surrounding have an impact on EVs:EVs interactions, which affect the uptake of NPCE EVs by TM cells.