Validation of an LDH assay for assessing nanoparticle toxicity

Abstract

Studies showed that certain cytotoxicity assays were not suitable for assessing nanoparticle (NP) toxicity. We evaluated a lactate dehydrogenase (LDH) assay for assessing copper (Cu-40, 40 nm), silver (Ag-35, 35 nm; Ag-40, 40 nm), and titanium dioxide (TiO2-25, 25 nm) NPs by examining their potential to inactivate LDH and interference with β-nicotinamide adenine dinucleotide (NADH), a substrate for the assay. We also performed a dissolution assay for some of the NPs. We found that the copper NPs, because of their high dissolution rate, could interfere with the LDH assay by inactivating LDH. Ag-35 could also inactivate LDH probably because of the carbon matrix used to cage the particles during synthesis. TiO2-25 NPs were found to adsorb LDH molecules. In conclusion, NP interference with the LDH assay depends on the type of NPs and the suitability of the assay for assessing NP toxicity should be examined case by case.

Minimal Intestinal Epithelial Cell Toxicity in Response to Short- and Long-Term Food-Relevant Inorganic Nanoparticle Exposure

“…44 TiO2 beyond loading levels of 100 µg/mL were found to adsorb on LDH. 45 SiO2 has been reported to interfere with the MTT assay in HeLa cells by promoting exocytosis of the formazan crystals, even at loading levels of 10 µg/mL. 46 Because of these literature reports, the experiments in the present study were conducted at a dose of 10 µg/cm 2 .…”

“…[44][45][46] In particular, for the MTT and LDH assays, it was suggested that loading levels be kept below 50 µg/cm 2 . With ZnO at a loading level of ~ 10 µg/cm 2 , slight effects were even observed with the LDH assay, but not with the MTT assay.…”

Development of biodegradable methylprednisolone microparticles for treatment of articular pathology using a spray-drying technique

“…31 Among the most widely used are the MTT assay which determines mitochondrial function in the cell 32 and the lactate dehydrogenase release assay which identifies cell membrane damage 33 or death. 34,35 Some researchers have shown that microparticles are able to induce an inflammatory response (demonstrated by release of interleukin-1β) because of the presence of inflammatory precursors, eg, bacteria or endotoxins acquired during the manufacturing process, certain components of the microparticle formulation (eg, polymers, excipients), and wastes produced by the manufacturing process (eg, organic solvents). 31 In vitro quantification of interleukin-1β is done using the enzymelinked immunosorbent assay, which has become the most frequently used method for quantification of interleukin-1β in supernatants of cell cultures via antibody and enzymatic recognition reactions.…”

Evaluation of Viability and Proliferation Profiles on Macrophages Treated with Silica Nanoparticles In Vitro via Plate-Based, Flow Cytometry, and Coulter Counter Assays

“…Some of NP properties that were reported to interfere with viability plate-based assays are surface charge, agglomeration/aggregation, hydrophobicity, and optical or magnetic properties [15][16][17]. For example, it was reported that titanium dioxide NPs (TiO 2 NP; various size and concentrations) bind lactate dehydrogenase (LDH, indicator of cell viability) and alter assay readout [18]; copper NPs (Cu NP, 40 nm) and silver NPs (Ag NP, 35 nm) inactivate LDH [19]; gold NPs (Au NP; 10 nm) can absorb and traffic amine-containing dyes inside cells resulting in false positive results for membrane permeability assays [11]; single-wall carbon nanotubes (SWCNTs) interact with and alter the readout of WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium), Coomasie Blue, Alamar Blue, Neutral Red, MTT, and plate-based cytotoxicity assays [20].…”