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

Journal Article · · PLoS ONE
 [1];  [2];  [2];  [3];  [3];  [3];  [2]; ORCiD logo [3];  [4]
  1. Charles Univ., Prague (Czech Republic); General University Hospital, Prague (Czech Republic); Academy of Sciences of the Czech Republic, Prague (Czech Republic); OSTI
  2. U.S. Food and Drug Administration (FDA), Silver Spring, MD (United States)
  3. Charles Univ., Prague (Czech Republic); General University Hospital, Prague (Czech Republic)
  4. West Virginia Univ., Morgantown, WV (United States)
+ Show Author Affiliations

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.

Research Organization:
Oak Ridge Inst. for Science and Education (ORISE), Oak Ridge, TN (United States)
Sponsoring Organization:
Czech Ministry of Education, Youth and Sports; Grant Agency of the Czech Republic; USDOE Office of Science (SC)
Grant/Contract Number:
SC0014664
OSTI ID:
1904917
Journal Information:
PLoS ONE, Journal Name: PLoS ONE Journal Issue: 10 Vol. 13; ISSN 1932-6203
Publisher:
Public Library of ScienceCopyright Statement
Country of Publication:
United States
Language:
English

References (42)

Apoptotic endothelial cells demonstrate increased adhesiveness for human mesenchymal stem cells journal April 2009
Interference of engineered nanoparticles with in vitro toxicity assays journal March 2012
Length reduction of multi-walled carbon nanotubes via high energy ultrasonication and its effect on their dispersibility journal July 2014
Multifunctional nanocarriers☆ journal December 2006
Spectroscopic analysis confirms the interactions between single walled carbon nanotubes and various dyes commonly used to assess cytotoxicity journal June 2007
A surface-grafted ligand functionalization strategy for coordinate binding of doxorubicin at surface of PEGylated mesoporous silica nanoparticles: Toward pH-responsive drug delivery journal January 2017
In vivo tumor targeting of tumor necrosis factor-α-loaded stealth nanoparticles: Effect of MePEG molecular weight and particle size journal January 2006
Biodegradable star HPMA polymer–drug conjugates: Biodegradability, distribution and anti-tumor efficacy journal September 2011
Photothermal ablation of tumor cells using a single-walled carbon nanotube–peptide composite journal January 2014
Nanotechnology for neurodegenerative disorders journal September 2012
Toxicity of carboxylated carbon nanotubes in endothelial cells is attenuated by stimulation of the autophagic flux with the release of nanomaterial in autophagic vesicles journal July 2014
Odyssey of a cancer nanoparticle: From injection site to site of action journal December 2012
Inactivation of lactate dehydrogenase by several chemicals: Implications for in vitro toxicology studies journal February 2007
Functionalized Silica Nanoparticles As an Alternative Platform for Targeted Drug-Delivery of Water Insoluble Drugs journal March 2016
Carbon Nanotubes in Electron Donor−Acceptor Nanocomposites journal November 2005
Preparation of Aligned Carbon Nanotubes with Prescribed Dimensions:  Template Synthesis and Sonication Cutting Approach journal April 2002
Surface Modification of Silica Nanoparticles to Reduce Aggregation and Nonspecific Binding journal April 2006
Oops They Did It Again! Carbon Nanotubes Hoax Scientists in Viability Assays journal April 2006
Carbon Black Nanoparticles Impair Acetylation of Aromatic Amine Carcinogens through Inactivation of Arylamine N-Acetyltransferase Enzymes journal May 2011
High-Throughput Screening Platform for Engineered Nanoparticle-Mediated Genotoxicity Using CometChip Technology journal March 2014
Necroptosis, necrosis and secondary necrosis converge on similar cellular disintegration features journal December 2009
NIH Image to ImageJ: 25 years of image analysis journal June 2012
Neutral red uptake assay for the estimation of cell viability/cytotoxicity journal June 2008
Cancer nanomedicine: progress, challenges and opportunities journal November 2016
Cell death assays for drug discovery journal March 2011
Staurosporine-induced apoptosis and hydrogen peroxide-induced necrosis in two human breast cell lines journal January 2003
The selection between apoptosis and necrosis is differentially regulated in hydrogen peroxide-treated and glutathione-depleted human promonocytic cells journal July 2003
A water-soluble tetrazolium salt useful for colorimetric cell viability assay journal January 1999
Nanomedicine in GI journal March 2011
In VitroMorphological Assessment of Apoptosis Induced by Antiproliferative Constituents from the Rhizomes ofCurcuma zedoaria
  • Syed Abdul Rahman, Syarifah Nur; Abdul Wahab, Norhanom; Abd Malek, Sri Nurestri
  • Evidence-Based Complementary and Alternative Medicine, Vol. 2013 https://doi.org/10.1155/2013/257108
journal January 2013
Nanotechnology in drug delivery: the need for more cell culture based studies in screening journal July 2014
The agglomeration state of nanoparticles can influence the mechanism of their cellular internalisation journal June 2017
Interlaboratory Evaluation of in Vitro Cytotoxicity and Inflammatory Responses to Engineered Nanomaterials: The NIEHS Nano GO Consortium journal June 2013
The Comparison of MTT and CVS Assays for the Assessment of Anticancer Agent Interactions journal May 2016
Toxicological considerations when creating nanoparticle-based drugs and drug delivery systems journal November 2011
Experimental considerations on the cytotoxicity of nanoparticles journal July 2011
Staurosporine induces different cell death forms in cultured rat astrocytes journal January 2012
Toxicity screenings of nanomaterials: challenges due to interference with assay processes and components of classicin vitrotests journal July 2013
Surface Modified Multifunctional and Stimuli Responsive Nanoparticles for Drug Targeting: Current Status and Uses journal August 2016
Targeted drug delivery to the brain using magnetic nanoparticles journal October 2015
The Unreliability of MTT Assay in the Cytotoxic Test of Primary Cultured Glioblastoma Cells journal September 2015
The agglomeration state of nanoparticles can influence the mechanism of their cellular internalisation collection January 2017

Similar Records

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
apoptosis
carbon nanotubes
cell enumeration techniques
fluorescence imaging
hydrogen peroxide
necrosis
toxicity
viable cell counting