Evaluation of Impedance-Based Label-Free Technology as a Tool for Pharmacology and Toxicology Investigations

Atienzar, Franck; Gerets, Helga; Tilmant, Karen; Toussaint, G; Dhalluin, Stéphane

doi:10.3390/bios3010132

Cited by 40 publications

(34 citation statements)

References 26 publications

(59 reference statements)

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“…This impedance-based label-free technology is noninvasive and allows the performance of kinetics measurements (11,12,27). Due to intrinsic growth characteristics, the cell density and the culture time period required to reach an appropriate growth phase at which time cells can be infected with the different viruses should be optimized (28).…”

Section: Discussionmentioning

confidence: 99%

Novel Method Based on Real-Time Cell Analysis for Drug Susceptibility Testing of Herpes Simplex Virus and Human Cytomegalovirus

2016

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The plaque reduction assay (PRA) is the gold standard phenotypic method to determine herpes simplex virus (HSV) and human cytomegalovirus (HCMV) susceptibilities to antiviral drugs. However, this assay is subjective and labor intensive. Here, we describe a novel antiviral phenotypic method based on real-time cell analysis (RTCA) that measures electronic impedance over time. The effective drug concentrations that reduced by 50% (EC 50 s) the cytopathic effects induced by HSV-1 and HCMV were evaluated by both methods. The EC 50 s of acyclovir and foscarnet against a reference wild-type ( H erpes simplex viruses 1 (HSV-1) and 2 (HSV-2) cause orolabial and genital infections as well as keratitis, encephalitis, and neonatal infections. Human cytomegalovirus (HCMV) is responsible for mononucleosis-like syndromes and organ-specific diseases in immunocompromised patients. All antiviral agents currently approved for the treatment of HSV and HCMV infections ultimately target the viral DNA polymerase (1). First-line antiviral agents for the treatment of HSV and HCMV infections include the nucleoside analogues acyclovir (ACV) and ganciclovir (GCV), respectively. Both drugs require a first phosphorylation by the thymidine kinase (TK) encoded by the UL23 gene (HSV) or the phosphotransferase encoded by the UL97 gene (HCMV) and two subsequent phosphorylations by cellular kinases to be converted into their active forms (2-4). The triphosphate forms compete with dGTP for incorporation into replicating DNA. Acyclovir triphosphate acts as a DNA chain terminator to inhibit viral replication, whereas ganciclovir triphosphate slows down DNA polymerization and eventually stops chain elongation. The pyrophosphate analogue foscarnet (FOS) is a second-line antiviral drug for the treatment of HCMV diseases and may also be used in the treatment of infections caused by nucleoside analogue-resistant HSV mutants. Foscarnet does not require any phosphorylation to be active (5). It directly inhibits the activity of the viral DNA polymerases encoded by UL30 (HSV) and UL54 (HCMV) genes. Foscarnet binds to the pyrophosphate binding site and blocks the release of pyrophosphate from the last nucleoside triphosphate added onto the growing DNA chain. Prolonged treatment with nucleoside analogues may be required to prevent or to manage HSV/HCMV infections in the immunocompromised host. Such prolonged antiviral therapy may result in the selection of viral isolates with reduced drug susceptibilities (6, 7).The plaque reduction assay (PRA) is the gold standard phenotypic method to determine the susceptibilities of HSV isolates to antiviral drugs and is approved as a standard protocol by the Clinical and Laboratory Standards Institute (8). The PRA has also been standardized in a consensus protocol for HCMV to decrease high interassay and interlaboratory variabilities (9). In this assay, cells are infected with a constant viral inoculum. The virus is then allowed to grow in the presence of serial drug dilutions for 2 to 3 (HSV) to 7 to 8 (HCMV) days before t...

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Section: Discussionmentioning

confidence: 99%

Novel Method Based on Real-Time Cell Analysis for Drug Susceptibility Testing of Herpes Simplex Virus and Human Cytomegalovirus

2016

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“…For studying kinetics, we used real-time cell impedance technology (RTCA). The CI reflects modifications of both cell morphology and cell viability (Atienzar et al 2013). HepaRG cells were exposed to 16 and 80 mg/cm 2 pristine, PEG, and DMPC M-MSNs for 3 days (Figure 3).…”

Section: Biocompatibility At Low Dosesmentioning

confidence: 99%

Biocompatibility assessment of functionalized magnetic mesoporous silica nanoparticles in human HepaRG cells

et al. 2017

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Magnetic mesoporous silica nanoparticles (M-MSNs) are a promising class of nanoparticles for drug delivery. However, a deep understanding of the toxicological mechanisms of action of these nanocarriers is essential, especially in the liver. The potential toxicity on HepaRG cells of pristine, pegylated (PEG), and lipid (DMPC) M-MSNs were compared. Based on MTT assay and real-time cell impedance, none of these NPs presented an extensive toxicity on hepatic cells. However, we observed by transmission electron microscopy (TEM) that the DMPC and pristine M-MSNs were greatly internalized. In comparison, PEG M-MSNs showed a slower cellular uptake. Whole gene expression profiling revealed the M-MSNs molecular modes of action in a time-and dose-dependent manner. The lowest dose tested (1.6 mg/cm 2 ) induced no molecular effect and was defined as 'No Observed Transcriptional Effect level.' The dose 16 mg/cm 2 revealed nascent but transient effects. At the highest dose (80 mg/cm 2 ), adverse effects have clearly arisen and increased over time. The limit of biocompatibility for HepaRG cells could be set at 16 mg/cm 2 for these NPs. Thanks to a comparative pathway-driven analysis, we highlighted the sequence of events that leads to the disruption of hepatobiliary system, elicited by the three types of MMSNs, at the highest dose. The Adverse Outcome Pathway of hepatic cholestasis was implicated. Toxicogenomics applied to cell cultures is an effective tool to characterize and compare the modes of action of many substances. We propose this strategy as an asset for upstream selection of the safest nanocarriers in the framework of regulation for nanobiosafety. ARTICLE HISTORY

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“…The medium was renewed every 2-3 days. The obtained differentiated HepaRG cells were seeded at a density of 1×10 5 cells/well in 96-well plates and employed for the cytotoxicity assay (MTT) as mentioned in a previous study 34 with slight modifications. Briefly, cells were incubated with the formulated flavonosomes at various concentrations ranging from 3.125 µg/mL to 200 µg/mL dissolved in culture medium and incubated for 24 hours.…”

Section: Cell Culture and Cytotoxicity Assaymentioning

confidence: 99%

Optimization, formulation, and characterization of multiflavonoids-loaded flavanosome by bulk or sequential technique

Karthivashan¹,

Kura²,

Abas³

et al. 2016

IJN

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This study involves adaptation of bulk or sequential technique to load multiple flavonoids in a single phytosome, which can be termed as “flavonosome”. Three widely established and therapeutically valuable flavonoids, such as quercetin (Q), kaempferol (K), and apigenin (A), were quantified in the ethyl acetate fraction of Moringa oleifera leaves extract and were commercially obtained and incorporated in a single flavonosome (QKA–phosphatidylcholine) through four different methods of synthesis – bulk (M1) and serialized (M2) co-sonication and bulk (M3) and sequential (M4) co-loading. The study also established an optimal formulation method based on screening the synthesized flavonosomes with respect to their size, charge, polydispersity index, morphology, drug–carrier interaction, antioxidant potential through in vitro 1,1-diphenyl-2-picrylhydrazyl kinetics, and cytotoxicity evaluation against human hepatoma cell line (HepaRG). Furthermore, entrapment and loading efficiency of flavonoids in the optimal flavonosome have been identified. Among the four synthesis methods, sequential loading technique has been optimized as the best method for the synthesis of QKA–phosphatidylcholine flavonosome, which revealed an average diameter of 375.93±33.61 nm, with a zeta potential of −39.07±3.55 mV, and the entrapment efficiency was >98% for all the flavonoids, whereas the drug-loading capacity of Q, K, and A was 31.63%±0.17%, 34.51%±2.07%, and 31.79%±0.01%, respectively. The in vitro 1,1-diphenyl-2-picrylhydrazyl kinetics of the flavonoids indirectly depicts the release kinetic behavior of the flavonoids from the carrier. The QKA-loaded flavonosome had no indication of toxicity toward human hepatoma cell line as shown by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide result, wherein even at the higher concentration of 200 µg/mL, the flavonosomes exert >85% of cell viability. These results suggest that sequential loading technique may be a promising nanodrug delivery system for loading multiflavonoids in a single entity with sustained activity as an antioxidant, hepatoprotective, and hepatosupplement candidate.

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Evaluation of Impedance-Based Label-Free Technology as a Tool for Pharmacology and Toxicology Investigations

Cited by 40 publications

References 26 publications

Novel Method Based on Real-Time Cell Analysis for Drug Susceptibility Testing of Herpes Simplex Virus and Human Cytomegalovirus

Novel Method Based on Real-Time Cell Analysis for Drug Susceptibility Testing of Herpes Simplex Virus and Human Cytomegalovirus

Biocompatibility assessment of functionalized magnetic mesoporous silica nanoparticles in human HepaRG cells

Optimization, formulation, and characterization of multiflavonoids-loaded flavanosome by bulk or sequential technique

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