Incompatibility of silver nanoparticles with lactate dehydrogenase leakage assay for cellular viability test is attributed to protein binding and reactive oxygen species generation.
A growing number of studies report that conventional cytotoxicity assays are incompatible with certain nanoparticles (NPs) due to artifacts caused by the distinctive characteristics of NPs. Lactate dehydrogenase (LDH) leakage assays have inadequately detected cytotoxicity of silver nanoparticles (AgNPs), leading to research into the underlying mechanism. When ECV304 endothelial-like umbilical cells were treated with citrate-capped AgNPs (cAgNPs) or bare AgNPs (bAgNPs), the plasma membrane was disrupted, but the LDH leakage assay failed to detect cytotoxicity, indicating interference with the assay by AgNPs. Both cAgNPs and bAgNPs inactivated LDH directly when treated to cell lysate as expected. AgNPs adsorbed LDH and thus LDH, together with AgNPs, was removed from assay reactants during sample preparation, with a resultant underestimation of LDH leakage from cells. cAgNPs, but not bAgNPs, generated reactive oxygen species (ROS), which were successfully scavenged by N-acetylcysteine or ascorbic acid. LDH inhibition by cAgNPs could be restored partially by simultaneous treatment with those antioxidants, suggesting the contribution of ROS to LDH inactivation. Additionally, the composition of the protein corona surrounding AgNPs was identified employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. In sum, the LDH leakage assay, a conventional cell viability test method, should be employed with caution when assessing cytotoxicity of AgNPs.
College of Pharmacy, Dongguk University, Goyang, Gyeonggi-do 410-820, Republic of Korea.
Seok-Jeong
SJ
Oh
College of Pharmacy, Dongguk University, Goyang, Gyeonggi-do 410-820, Republic of Korea.
Hwa
H
Kim
College of Pharmacy, Dongguk University, Goyang, Gyeonggi-do 410-820, Republic of Korea.
Yingqiu
Y
Liu
College of Pharmacy, Dongguk University, Goyang, Gyeonggi-do 410-820, Republic of Korea.
Hyo-Kyung
HK
Han
College of Pharmacy, Dongguk University, Goyang, Gyeonggi-do 410-820, Republic of Korea.
Kyenghee
K
Kwon
College of Pharmacy, Dongguk University, Goyang, Gyeonggi-do 410-820, Republic of Korea.
Kyung-Hwa
KH
Chang
College of Pharmacy, Dongduk Woman's University, Seoul 136-714, Republic of Korea.
Kwangsik
K
Park
Department of Chemical Engineering, Kwangwoon University, Seoul 139-701, Republic of Korea.
Younghun
Y
Kim
Department of Bionano Technology, Gachon Bionano Research Institute, Gachon University, Sungnam, Gyeonggi-do 461-701, Republic of Korea.
Kyuhwan
K
Shim
Department of Bionano Technology, Gachon Bionano Research Institute, Gachon University, Sungnam, Gyeonggi-do 461-701, Republic of Korea.
Seong Soo A
SS
An
College of Pharmacy, Dongguk University, Goyang, Gyeonggi-do 410-820, Republic of Korea. Electronic address: mlee@dongguk.edu.
Moo-Yeol
MY
Lee
D000111:Acetylcysteine / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects*; D002853:Chromatography, Liquid; D005562:Formazans / Q000032:analysis; D006367:HeLa Cells; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000032:analysis*; D053768:Metal Nanoparticles / Q000648:ultrastructure*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012834:Silver / Q000633:toxicity*; D021241:Spectrometry, Mass, Electrospray Ionization; D013778:Tetrazolium Salts / Q000032:analysis
D005562:Formazans; D017382:Reactive Oxygen Species; D013778:Tetrazolium Salts; C079782:MTT formazan; D012834:Silver; D007770:L-Lactate Dehydrogenase; D000111:Acetylcysteine
Cytotoxicity; Lactate dehydrogenase leakage assay; Protein adsorption; Reactive oxygen species; Silver nanoparticles