An effective “three-in-one” screening assay for testing drug and nanoparticle toxicity in human endothelial cells

Abstract

Evaluating nanoparticle (NP) toxicity in human cell systems is a fundamental requirement for future NP biomedical applications. In this study, we have designed a screening assay for assessing different types of cell death induced by NPs in human umbilical vein endothelial cell (HUVEC) culture. This assay consists of WST-8, LDH and Hoechst 33342 staining, all performed in one well, which enables an evaluation of cell viability, necrosis and apoptosis, respectively, in the same cell sample. The 96-well format and automated processing of fluorescent images enhances the assay rapidity and reproducibility. After testing the assay functionality with agents that induced different types of cell death, we investigated the endothelial toxicity of superparamagnetic iron oxide nanoparticles (SPIONs, 8 nm), silica nanoparticles (SiNPs, 7–14 nm) and carboxylated multiwall carbon nanotubes (CNTCOOHs, 60 nm). Our results indicated that all the tested NP types induced decreases in cell viability after 24 hours at a concentration of 100 μg/ml. SPIONs caused the lowest toxicity in HUVECs. By contrast, SiNPs induced pronounced necrosis and apoptosis. A time course experiment showed the gradual toxic effect of all the tested NPs. CNTCOOHs inhibited tetrazolium derivatives at 100 μg/ml, causing false negative results from the WST-8 and LDH assay. In summary, our data demonstrate that the presented “three-in-one” screening assay is capable of evaluating NP toxicity effectively and reliably. Due to its simultaneous utilization of two different methods to assess cell viability, this assay is also capable of revealing, if NPs interfere with tetrazolium salts.

Induced pluripotent stem cell-derived vascular networks to screen nano–bio interactions

“…32 Zinc oxide NPs were observed to be toxic (450%) at 50 mg mL À1 in human aortic ECs, 34 human cardiac microvascular ECs 35 and in HUVECs; 36,37 all these results are in good accordance with the 60% toxicity observed in our study for ECs derived from iPSCs. SiO 2 NPs have also been reported to induce toxicities from 20 to 60% in HUVECs for NP concentrations between 50 and 75 mg mL À1 , 38 while Ag NPs have been reported to induce toxicity in rat brain microvessel ECs 49 and to demonstrate anti-angiogenic properties. 50 It is possible that the higher toxicity of Ag NPs against HGPS-iPSC ECs, as compared to that of N-iPSC ECs, is related to a decrease in the antioxidative stress response program in HGPS-iPSC ECs.…”

“…36,37 Regarding NP28, the results are also in accordance with those reported in the literature for HUVECs exposed for 24 h, with toxicities ranging from 20 to 60% for 50 and 75 mg mL À1 SiO 2 concentrations, respectively. 38 NPs showed a differential toxic effect against both types of ECs. HGPS-iPSC ECs were more sensitive to NP13, NP18, NP22, NP26 and NP29 NPs as compared to N-iPSC ECs (Fig.…”

Targeting Tunable Physical Properties of Materials for Chronic Wound Care

“…More importantly, ZnO NPs significantly increased intracellular ROS showing a stronger cellular cytotoxicity than that of Ag and TiO 2 NPs. In another case, Filipova et al (2018) reported a "three-in-one" screening assay to test the toxicity of three kinds of NPs for human umbilical vein endothelial cells. The three NPs are silica NPs (7-14 nm), superparamagnetic iron oxide NPs (8 nm), and carboxylated multiwall carbon nanotubes (60 nm), all of which were tested at the same concentration of 100 µg/ml.…”

Interference: A Much-Neglected Aspect in High-Throughput Screening of Nanoparticles

“…Their HTS setup included assays for cellular membrane permeability by propidium iodide (PI) uptake, intracellular calcium flux by Fluo4 fluorophore, reactive oxygen species (ROS) production by MitoSox Red fluorophore, and mitochondrial membrane potential by 5,5 0 ,6,6 0 -tetrachloro-1,1 0 ,3,3 0 -tetraethyl-benzamidazolocarbocyanin iodide (JC-1) fluorophore. 26 More recently, Filipova et al 27 suggested a "three-in-one" screening assay for NP toxicity, which could be scaled up for high-throughput testing. Their screening assay, however, consisted of the WST-8 tetrazolium salt assay, LDH assay, and Hoechst 33342, of which the WST-8 and LDH assays showed interference with carbon-based NPs.…”

“…Their screening assay, however, consisted of the WST-8 tetrazolium salt assay, LDH assay, and Hoechst 33342, of which the WST-8 and LDH assays showed interference with carbon-based NPs. 27 It would have been valuable if the authors had assessed all types of interference mechanisms present instead of just only one mechanism. Several other HTS screening programs have also been devised consisting of the DNA intercalating dye, Hoechst 33342, to assess cell number and nuclear size, JC-1 dye to assess perturbation of mitochondrial membrane potential, Fluo4, and PI.…”