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Nano and Micro Technologies

Nano and Micro Technologies - Poster Session D

Poster Y20 - Altered K+ and Ca2+ Flux in Endothelial Cells upon Exposure to 20 nm Nanoparticles

Friday, October 25, 2024
3:30 PM – 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  In the United States, stroke is the fifth leading cause of death, and 87% of strokes are ischemic. One promising method of treating ischemic stroke is the use of vasodilators, medications that induce blood vessel widening to allow more blood flow through the constricted vessel or collateral vessels while also lowering blood pressure. At a cellular level, vasodilation involves an influx of calcium in endothelial cells, triggering hyperpolarization of the endothelial and smooth muscle membranes through the corresponding outflux of potassium. Our recent work demonstrates that inorganic nanoparticles within a small size range can induce calcium uptake in the endothelium. Specifically, particles of 20 nm diameter made of gold trigger calcium influx via the action of the transient receptor potential vanilloid-type 4 (TRPV4) cation channel. While this unique mechanism carries potential for therapeutic application, there is limited current knowledge on whether 20 nm nanoparticles cause vasodilation in vivo and the mechanism by which these particles trigger the action of TRPV4. The work presented here begins to address this gap in knowledge by studying the impact of 20 nm gold nanoparticles (AuNPs) on the influx of calcium and activation of potassium channels in cultured endothelial cells.

Materials and

Methods:  In order to examine the flux of calcium and potassium, the fluorescent dyes Fluoforte (Enzo Life Sciences) and Ion Potassium Green 4 AM (IPG-4, ION Biosciences) were used to examine calcium and potassium respectively in Human umbilical vein endothelial cells (HUVEC, Lonza) grown in static culture. HUVEC were grown under standard conditions and exposed to concentrations of 5, 10, 20, 40, 60, 80, 100, 150, 200, and 300 µg/ml of AuNPs (Sigma) stabilized in 0.1 mM phosphate buffer saline (PBS) for 2 and 24 Hours. Controls of untreated cells and 0.1 mM PBS equal to the concentration of AuNPs were used. Qualitative images were taken on an EVOS 5000 fluorescence microscope and quantitative fluorescence was measured by SpectraMax i3x Plate reader. Fluorescent Images were evaluated in ImageJ to obtain the corrected total cell fluorescence (CTCF) by measuring the integrated density, area, and means of collected images and using the following formula to calculate CTCF: Average Integrated Density – (Average Area of the Image * Average Mean of the Blanks). Cell counting kit 8 (CCK-8) was used to measure viability of HUVEC exposed to the same concentrations of AuNPs and PBS. Fluorescence values were normalized by cell viability to obtain a final value. All studies were done in quadruplicate and statistically evaluated using two-way ANOVA and post-hoc t-tests.

Results, Conclusions, and Discussions: Similar to trends seen in previous studies, our results at 2 hours of AuNP exposure indicate that 20 nm AuNPs induce an influx of calcium and outflux of potassium as shown in Figure 1 compared to untreated cells.1 However, at 24 hours, the flux of these ions switched with concentrations of 40, 60, 80, 100, 150, 200, and 300 µg/mL showing a negative percent change in calcium compared to untreated cells, indicating an outflux of calcium that was maximized at -272% at the highest AuNP concentration. A similar effect was observed in potassium flux at 24 hours with concentrations of 5, 20, 40, 60, 80, 100, 200, and 300 µg/mL displaying a positive percent change compared to untreated cells, indicating an influx of potassium that was maximized at 984% at the highest AuNP concentration. These variations between 2 and 24 hours indicate a potential return to homeostasis over time similar to what has been previously observed in HUVEC after exposure to 20 nm AuNPs.2 Statistical analysis revealed significant results at both 2 and 24 hours as shown in Figure 1. Since results reveal that concentration-matched PBS affects calcium and potassium flux, our planned studies will seek to eliminate this confounding affect by concentrating gold nanoparticles to remove PBS before introduction to the cells. A dynamic study to examine the time-dependence of these effects will also be conducted. In conclusion, 20 nm AuNPs induce a flux of calcium and potassium within endothelial cells, yet the effect of the solvent PBS requires further refinement of experimental methodology.

Acknowledgements (Optional):  Funding for this study as well as financial support for the lead author were provided by Vermont Space Grant Consortium Graduate Fellowship.