Determination of endogenous trace elements in extracellular vesicles secreted by an in vitro model of human retinal pigment epithelium under oxidative stress conditions using ICP-MS

The determination of endogenous Fe, Cu and Zn in exosomes (<200 nm extracellular vesicles) secreted by an in vitro model of the human retinal pigment epithelium (HRPEsv cell line) was carried out by inductively coupled plasma - mass spectrometry (ICP-MS). Results for cells treated with 2,2′-azobis (2-methylpropionamidine) dihydrochloride (AAPH) inducing oxidative stress (OS) conditions were compared with non-treated (control) cells in order to evaluate possible differences in the metal composition between both groups. Three sample introduction systems were tested for ICP-MS analysis: a micronebulizer and two single cell nebulization systems (as total consumption set-ups), being found one of the single cell systems (operating in bulk mode) as the most suitable. Two protocols for the isolation of exosomes from cell culture media were investigated based on differential centrifugation and precipitation with a polymer-based reagent. Transmission electron microscopy measurements showed smaller and more homogeneous sizes (15–50 nm versus 20–180 nm size range) together with a higher particle concentration for exosomes purified by precipitation compared to differential centrifugation. However, it was observed that the contribution of polymer-based protocol to the Fe, Cu and Zn blank was significant as compared to the differential centrifugation protocol. Therefore, considering the low concentrations of the evaluated endogenous elements in exosomes from the HRPEsv cell line, the polymer-based precipitation method was discarded. When comparing metal levels in samples from control versus OS-treated HRPEsv cells, results for Fe and Cu were statistically similar. However, upregulation of Zn was found during OS conditions (11 versus 34 μg L in control and OS-treatment, respectively), showing Zn depletion through secretory activity induced by OS, underlying the antioxidant ability of RPE cells.

This work was financially supported through projects PID2019-107838RB-I00/Agencia Estatal de Investigación (AEI)/10.13039/501100011033) and the PCTI Program of the Government of the Principality of Asturias and FEDER Program of the European Union (No. AYUD/2021/51289). J. Martínez-García acknowledges the “Severo Ochoa” Program Grant with Ref. BP21-041 (Principality of Asturias, Spain). Authors want to acknowledge C. Derrick Quarles Jr. for his support with the microFAST Single Cell system, and the technical support provided by Servicios Científico-Técnicos of the University of Oviedo – Mass Spectrometry and Electron Microscopy Units. The Instituto Oftalmológico Fernández-Vega and Fundación de Investigación Oftalmológica acknowledge financial support from the Fundación Rafael del Pino (http://www.frdelpino.es), through the “Cátedra Rafael del Pino” and from IDE/2019/000289 project of “Instituto de Desarrollo Económico del Principado de Asturias” (IDEPA) of Gobierno del Principado de Asturias and FEDER.