In Vitro Cytotoxicity Assessment

O’Brien, Peter J.; Haskins, Jeffrey R.

doi:10.1385/1-59745-217-3:415

Cited by 27 publications

(39 citation statements)

References 22 publications

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“…Although such assays are pertinent because some AMPs are thought to act exclusively through perturbation of phospholipid bilayers [8], subtle cytotoxicity should also be examined in mammalian cells in order to understand how AMPs act on a range of cellular processes in sub-lethal concentrations, and not just by their ability to cause cytolysis. This is important because determination of drug safety is multi-factorial and depends on maintenance of virtually all cellular events [9].…”

Section: Introductionmentioning

confidence: 99%

“…Unlike conventional cytotoxicity assays such as the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) conversion assay, which detects overt, gross cytotoxicity, HCA provides pre-lethal toxicity assays that have high sensitivity and specificity [9,19,21]. HCA combines flow cytometry with automated fluorescence microscopy and advanced image analysis software, permitting quantitative analysis of multiple cellular events in single cells (Fig.…”

Section: Introductionmentioning

confidence: 99%

“…Screening involved detecting changes in cell number (CN), nuclear area (NA), nuclear intensity (NI), mitochondrial membrane potential (MMP), plasma membrane permeability (PMP) and intracellular calcium (IC). These parameters measure selected cellular metabolic events and were optimised for HCA because they are hallmarks of cell health [9,19].…”

Section: Introductionmentioning

confidence: 99%

“…The duration of exposure was chosen to encompass acute and chronic exposure because 75% of molecules typically do not express acute cytotoxicity [9]. Human intestinal Caco-2 cells were selected for assessment because of current interest in use of AMPs both to treat enteric infections and as potential food additives.…”

Section: Introductionmentioning

confidence: 99%

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High content analysis to determine cytotoxicity of the antimicrobial peptide, melittin and selected structural analogs

et al. 2011

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Antimicrobial peptides (AMPs) are naturally occurring entities with potential as pharmaceutical candidates and/or food additives. They are present in many organisms including bacteria, insects, fish and mammals. While their antimicrobial activity is equipotent with many commercial antibiotics, current limitations are poor pharmacokinetics, stability and potential toxicology issues. Most elicit antimicrobial action via perturbation of bacterial membranes. Consequently, associated cytotoxicity in human cells is reflected by their capacity to lyse erythrocytes. However, more rigorous toxicological assessment of AMPs is required in order to predict potential failure at a later stage of development. We describe a high-content analysis (HCA) screening protocol recently established for determination and prediction of safety in pharmaceutical drug discovery. HCA is a powerful, multi-parameter bioanalytical tool that amalgamates the actions of fluorescence microscopy with automated cell analysis software in order to understand multiple changes in cellular health. We describe the application of HCA in assessing cytotoxicity of the cytolytic α-helical peptide, melittin, and selected structural analogues. The data shows that structural modification of melittin reduces its cytotoxic action and that HCA is suitable for rapidly identifying cytotoxicity.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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High content analysis to determine cytotoxicity of the antimicrobial peptide, melittin and selected structural analogs

et al. 2011

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“…Several cytotoxicity assays have been specifically developed for the assessment of apoptosis, including detection of plasma membrane annexin V, caspase activation, and shrinkage and fragmentation of nuclei. The implementation of sophisticated assays for screening during the 1990s resulted from the development and automation of multiwell, microtiter-plate high-content readers and the development of relatively nontoxic, subcellular, fluorescent dyes [17]. These sensitive assays screen overt and acute cytotoxicity and have a major role in drug discovery [13].…”

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confidence: 99%

High-content analysis for drug delivery and nanoparticle applications

Brayden

Cryan

Dawson

et al. 2015

Drug Discovery Today

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Publication information Drug Discovery Today, 20 (8): 942-957Publisher Elsevier Item record/more information http://hdl.handle.net/10197/6636 Publisher's statementThis is the author's version of a work that was accepted for publication in Drug Discovery Today. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Drug Discovery Today, 20 (8), (2015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Page 1 of 26A c c e p t e d M a n u s c r i p t Sally-Ann CryanSally-Ann Cryan has a pharmacy degree and a PhD in pharmaceutics ( Jeremy SimpsonJeremy Simpson carried out his PhD at the University of Warwick, followed postdoctoral work at the Scripps Research Institute in San Diego, and the Imperial Cancer Research Fund in London. After 9 years as a staff member at the European Molecular Biology Laboratory (EMBL) in Heidelberg (Germany), he was appointed as professor of cell biology at UCD, in 2008. He currently applies high-throughput imaging technologies to study subcellular transport pathways and the internalization routes taken by nanoparticles in cells. He runs the UCD Cell Screening Laboratory and is the author of more than 80 publications.High-content analysis (HCA) provides quantitative multiparametric cellular fluorescence data. From its origins in discovery toxicology, it is now addressing fundamental questions in drug delivery. Nanoparticles (NPs), polymers, and intestinal permeation enhancers are being harnessed in drug delivery systems to modulate plasma membrane properties and the intracellular environment. Identifying comparative mechanistic cytotoxicity on sublethal events is crucial to expedite the development of such systems. NP uptake and intracellular routing pathways are also being dissected using chemical and genetic perturbations, with the potential to assess the intracellular fate of targeted and untargeted particles in vitro. As we discuss here, HCA is set to make a major impact in preclinical delivery research by elucidating the intracellular pathways of NPs and the in vitro mechanistic-based toxicology of formulation constituents. A brief recap of cytotoxicity assaysIn vitro cell-based assays have long been used to assess the cytotoxic effects of drug exposure [1]. Cells undergoing acute necrosis swell and lose metabolic capacity, thereby losing the capacity to maintain a barrier to the extracellular space and the ability ...

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High‐Throughput Cytotoxicity Screening by Propidium Iodide Staining

et al. 2007

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This unit describes a system for the automated high‐throughput analysis of cell cytotoxicity in 96‐well and 384‐well microplates. Discrete cell cultures are analyzed at rates of 40/min (∼2.5 min/96 wells, ∼10 min/384 wells) and cytotoxicity is quantified on the basis of a combination of propidium iodide (PI) fluorescence analysis and cell counting performed by the flow cytometer. Only 2 µl is aspirated from a culture for analysis so that assays can be performed in small volumes to minimize reagent cost and usage.

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In Vitro Cytotoxicity Assessment

Cited by 27 publications

References 22 publications

High content analysis to determine cytotoxicity of the antimicrobial peptide, melittin and selected structural analogs

High content analysis to determine cytotoxicity of the antimicrobial peptide, melittin and selected structural analogs

High-content analysis for drug delivery and nanoparticle applications

High‐Throughput Cytotoxicity Screening by Propidium Iodide Staining

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