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Understanding and improving assays for cytotoxicity of nanoparticles: what really matters?

ABSTRACT: Despite years of excellent individual studies, the impact of nanoparticle (NP) cytotoxicity studies remains limited by inconsistent data collection and analysis. It is often unclear how exposure conditions can be used to determine cytotoxicity quantitatively. Discrepancies due to using different measurement conditions, readouts and controls to characterize NP interactions with cells lead to further challenges. To examine which parameters are critical in NP cytotoxicity studies, we have chosen to examine two NP types (liposomes and quantum dots) at different concentrations incubated with two primary vascular endothelial cells, HUVEC and HMVEC-C for a standard time of 24 h. We paid close attention to the effects of positive controls and cell association on interpretation of cytotoxicity data. Various cellular responses (ATP content, oxidative stress, mitochondrial toxicity, and phospholipidosis) were measured in parallel. Interestingly, cell association data varied significantly with the different image analyses. However, cytotoxicity responses could all be correlated with exposure concentration. Cell type did have an effect on cytotoxicity reports. Most significantly, NP cytotoxicity results varied with the inclusion or exclusion of positive controls. In the absence of positive controls, one tends to emphasize small changes in cell responses to NPs.

SUBMITTER: Labouta HI

PROVIDER: S-EPMC9081595 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Understanding and improving assays for cytotoxicity of nanoparticles: what really matters?

Labouta Hagar I HI Sarsons Christopher C Kennard Jacob J Gomez-Garcia M Juliana MJ Villar Kenrick K Lee Hyungbok H Cramb David T DT Rinker Kristina D KD

RSC advances 20180622 41

Despite years of excellent individual studies, the impact of nanoparticle (NP) cytotoxicity studies remains limited by inconsistent data collection and analysis. It is often unclear how exposure conditions can be used to determine cytotoxicity quantitatively. Discrepancies due to using different measurement conditions, readouts and controls to characterize NP interactions with cells lead to further challenges. To examine which parameters are critical in NP cytotoxicity studies, we have chosen to ...[more]

PMID: 35540163

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Project description:BackgroundBlack salve is a controversial complementary and alternative medicine (CAM) associated with skin toxicity and skin cancer treatment failures. Black salve formulations vary between manufacturers and contain a number of botanical and synthetic constituents. The skin cancer cytotoxicity of a number of these constituents has not been assessed to date. The alkaloids from the rhizomes of Sanguinaria canadensis, a key black salve ingredient, have had their single compound cytotoxicity assessed; however, whether they possess synergistic cytotoxicity with other compounds has not been studied and is of direct clinical relevance. This research aimed to improve our understanding of the skin cancer cytotoxicity of black salve constituents.MethodsThe cytotoxicity of individual and combination black salve constituents were assessed against the A375 melanoma and A431 squamous cell carcinoma cell lines. Cytotoxicity was determined using the Resazurin assay with fluorescence measured using a Tecan Infinite 200 Pro Microplate reader, compound cytotoxicity being compared to that of the topical cancer therapeutic agent, 5- fluouracil. Docetaxal was used as a positive control. Dunnetts p value was used to determine whether significant synergistic cytotoxicity was present.ResultsSanguinarine was the most cytotoxic compound tested with a 24-hour IC50 of 2.1 μM against the A375 Melanoma cell line and 3.14 μM against the A431 SCC cell line. All black salve constituents showed greater cytotoxicity against the two skin cancer cell lines tested than the skin cancer therapeutic 5-Fluouracil with 24 hours of compound exposure. Chelerythrine and minor Quaternary Benzophenanthridine Alkaloids (QBAs) present in black salve, at concentrations not having a cytotoxic effect by themselves, boosted the cytotoxic effects of sanguinarine. This could be a synergistic rather than additive cytotoxic effect although the synergistic effect was cell line and concentration dependent.ConclusionsBlack salve contains several cytotoxic compounds, a number of which have been found to possess synergistic cytotoxicity for the first time against skin cancer cell lines. In addition, these compounds together increase the overall cytotoxic effect. Assessing multi-compound cytotoxicity in herbal medicine can provide additional information about both their therapeutic and toxicity potential. As black salve is currently being used by patients, further cytotoxicity work should be undertaken to assess whether synergistic cytotoxicity exists when tested in normal skin cells.

Project description:BackgroundIron oxide magnetic nanoparticles (MNP's) have an increasing number of biomedical applications. As such in vitro characterisation is essential to ensure the bio-safety of these particles. Little is known on the cellular interaction or effect on membrane integrity upon exposure to these MNPs. Here we synthesised Fe(3)O(4) and surface coated with poly(ethylenimine) (PEI) and poly(ethylene glycol) (PEG) to achieve particles of varying surface positive charges and used them as model MNP's to evaluate the relative utility and limitations of cellular assays commonly applied for nanotoxicity assessment. An alternative approach, atomic force microscopy (AFM), was explored for the analysis of membrane structure and cell morphology upon interacting with the MNPs. The particles were tested in vitro on human SH-SY5Y, MCF-7 and U937 cell lines for reactive oxygen species (ROS) production and lipid peroxidation (LPO), LDH leakage and their overall cytotoxic effect. These results were compared with AFM topography imaging carried out on fixed cell lines.ResultsSuccessful particle synthesis and coating were characterised using FTIR, PCS, TEM and ICP. The particle size from TEM was 30 nm (-16.9 mV) which increased to 40 nm (+55.6 mV) upon coating with PEI and subsequently 50 nm (+31.2 mV) with PEG coating. Both particles showed excellent stability not only at neutral pH but also in acidic environment of pH 4.6 in the presence of sodium citrate. The higher surface charge MNP-PEI resulted in increased cytotoxic effect and ROS production on all cell lines compared with the MNP-PEI-PEG. In general the effect on the cell membrane integrity was observed only in SH-SY5Y and MCF-7 cells by MNP-PEI determined by LDH leakage and LPO production. AFM topography images showed consistently that both the highly charged MNP-PEI and the less charged MNP-PEI-PEG caused cell morphology changes possibly due to membrane disruption and cytoskeleton remodelling.ConclusionsOur findings indicate that common in vitro cell endpoint assays do not give detailed and complete information on cellular state and it is essential to explore novel approaches and carry out more in-depth studies to elucidate cellular response mechanism to magnetic nanoparticles.

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Understanding and improving assays for cytotoxicity of nanoparticles: what really matters?

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Understanding and improving assays for cytotoxicity of nanoparticles: what really matters?

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