Influence of Anti-Glaucoma Drugs on Uptake of Extracellular Vesicles by Trabecular Meshwork Cells

Abstract

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 incubated 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.

Non-Pigmented Ciliary Epithelium-Derived Extracellular Vesicles Loaded with SMAD7 siRNA Attenuate Wnt Signaling in Trabecular Meshwork Cells In Vitro

“…An imaging flow cytometer study used to track the kinetics of NPCE EVs internalization ratio by TM cells resulted in a maximal uptake ratio of EVs to TM cells after 24 h [ 9 ]. Since our previous data demonstrated that EVs are mediators of the NPCE-TM cells communication at early time points, in the present study we examined the effects of loaded EVs with siRNA on TM cells at an earlier time point of 24 h rather than 48 h, 72 h, or 96 h. Tracking the expression of Wnt-TGFβ2 proteins in TM cells exposed to NPCE EVs, led to a significant attenuation of the expression levels of all examined proteins ( Figure 6 ) as was previously shown [ 9 , 40 , 41 ]. Targeting SMAD7 in TM cells with electroporated EVs, eliminated the decrease of both β-Catenin ( Figure 6 D) and pGSK3 ( Figure 6 C) proteins levels observed upon addition NPCE EVs to TM cells.…”

“…In earlier studies in our lab, involving human NPCE-derived exosomes’ characterization [ 37 , 38 ], we were able to provide evidence for their role as signaling mediators of the Wnt signaling pathway in TM cells. These EVs demonstrated a specific uptake mechanism [ 39 ] influenced by particle interactions [ 40 , 41 ]. We suggested that these native biological nanoparticles mediate the communication between NPCE and TM cells by ECM remodeling [ 42 ], supporting a physiological role in IOP regulation.…”

Electrostatic Charge‐Mediated Apoptotic Vesicle Biodistribution Attenuates Sepsis by Switching Neutrophil NETosis to Apoptosis

“…Previous studies show the increase of zeta potential of EVs enhances its endocytosis by neighboring cell. [21,34] When neutrophils were treated with PMA, NETs generated, and released histone with positive charge. Liposomes (++) have repulsive force with histone.…”

Intravitreal Injectable Hydrogels for Sustained Drug Delivery in Glaucoma Treatment and Therapy

“…Eye drops are a fundamental type of topical administration due to their ease of application, favorable cost, and good patient consistency [35]. The typical retention time of eye drops administered topically in the pre-corneal tear film is about 1 min.…”