Limits...
Dilemmas in the reliable estimation of the in-vitro cell viability in magnetic nanoparticle engineering: which tests and what protocols?

Hoskins C, Wang L, Cheng WP, Cuschieri A - Nanoscale Res Lett (2012)

Bottom Line: Full understanding of the interactions between MNPs and mammalian cells is a critical issue for their applications.The Titer-Glo also experienced a small overestimation.To further understand the cytotoxic effect of the nanoparticles on these cells, reactive oxygen species production, lipid peroxidation and cell membrane integrity were investigated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Medical Science and Technology (IMSaT), Wilson House, 1 Wurzburg Loan, University of Dundee, Dundee, DD2 1FD, UK. l.y.wang@dundee.ac.uk.

ABSTRACT
Magnetic nanoparticles [MNPs] made from iron oxides have many applications in biomedicine. Full understanding of the interactions between MNPs and mammalian cells is a critical issue for their applications. In this study, MNPs were coated with poly(ethylenimine) [MNP-PEI] and poly(ethylene glycol) [MNP-PEI-PEG] to provide a subtle difference in their surface charge and their cytotoxicity which were analysed by three standard cell viability assays: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium [MTS], CellTiter-Blue and CellTiter-Glo (Promega, Southampton, UK) in SH-SY5Y and RAW 264.7 cells The data were validated by traditional trypan blue exclusion. In comparison to trypan blue manual counting, the MTS and Titer-Blue assays appeared to have consistently overestimated the viability. The Titer-Glo also experienced a small overestimation. We hypothesise that interactions were occurring between the assay systems and the nanoparticles, resulting in incorrect cell viability evaluation. To further understand the cytotoxic effect of the nanoparticles on these cells, reactive oxygen species production, lipid peroxidation and cell membrane integrity were investigated. After pegylation, the MNP-PEI-PEG possessed a lower positive surface charge and exhibited much improved biocompatibility compared to MNP-PEI, as demonstrated not only by a higher cell viability, but also by a markedly reduced oxidative stress and cell membrane damage. These findings highlight the importance of assay selection and of dissection of different cellular responses in in-vitro characterisation of nanostructures.

No MeSH data available.


Related in: MedlinePlus

Cell viability of SH-SY5Y and RAW 264.7 cells. A1 SH-SY5Y cells incubated with MNP-PEI nanoparticles, A2 RAW 264.7 cells incubated with MNP-PEI nanoparticles, B1 SH-SY5Y cells incubated with MNP-PEI-PEG nanoparticles and B2 RAW 264.7 cells incubated with MNP-PEI-PEG nanoparticles. The cells were incubated at different concentrations as indicated over a 72-h incubation. Cell viability was determined using common assays including MTS assay (square), CellTiter-Blue assay (triangle), CellTiter-Glo assay (circle) and trypan blue counting (diamond). (n = 3 ± SE). Asterisk denotes significantly increased level of cell viability compared with trypan blue measurement (p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3275459&req=5

Figure 2: Cell viability of SH-SY5Y and RAW 264.7 cells. A1 SH-SY5Y cells incubated with MNP-PEI nanoparticles, A2 RAW 264.7 cells incubated with MNP-PEI nanoparticles, B1 SH-SY5Y cells incubated with MNP-PEI-PEG nanoparticles and B2 RAW 264.7 cells incubated with MNP-PEI-PEG nanoparticles. The cells were incubated at different concentrations as indicated over a 72-h incubation. Cell viability was determined using common assays including MTS assay (square), CellTiter-Blue assay (triangle), CellTiter-Glo assay (circle) and trypan blue counting (diamond). (n = 3 ± SE). Asterisk denotes significantly increased level of cell viability compared with trypan blue measurement (p < 0.05).

Mentions: An interesting phenomenon was observed when measuring the cell viability of both the SH-SY5Y (Figure 2A1, B1) and RAW 264.7 (Figure 2A2, B2) cells after incubation with the MNPs using MTS, CellTiter-Blue and CellTiter-Glo assays. At increased concentrations, the MNPs became attached to either the cell membrane or the bottom of the plate, appearing as a brown colour in the well (even after five washes with fresh culture media) (data not shown). The presence of these MNPs resulted in greater absorption readings in the MTS assay and thus significantly showed an overestimation of the cell viability (p < 0.05). This phenomenon could be explained by the adherence of the sticky polymers to the well surface or the positive amine groups being attracted to the negative charge of the cell membrane. After pegylation, the interference appeared to have been reduced; however, the value of cell viability that appeared was still larger compared to the visual inspection of viable cells under microscope (data not shown).


Dilemmas in the reliable estimation of the in-vitro cell viability in magnetic nanoparticle engineering: which tests and what protocols?

Hoskins C, Wang L, Cheng WP, Cuschieri A - Nanoscale Res Lett (2012)

Cell viability of SH-SY5Y and RAW 264.7 cells. A1 SH-SY5Y cells incubated with MNP-PEI nanoparticles, A2 RAW 264.7 cells incubated with MNP-PEI nanoparticles, B1 SH-SY5Y cells incubated with MNP-PEI-PEG nanoparticles and B2 RAW 264.7 cells incubated with MNP-PEI-PEG nanoparticles. The cells were incubated at different concentrations as indicated over a 72-h incubation. Cell viability was determined using common assays including MTS assay (square), CellTiter-Blue assay (triangle), CellTiter-Glo assay (circle) and trypan blue counting (diamond). (n = 3 ± SE). Asterisk denotes significantly increased level of cell viability compared with trypan blue measurement (p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3275459&req=5

Figure 2: Cell viability of SH-SY5Y and RAW 264.7 cells. A1 SH-SY5Y cells incubated with MNP-PEI nanoparticles, A2 RAW 264.7 cells incubated with MNP-PEI nanoparticles, B1 SH-SY5Y cells incubated with MNP-PEI-PEG nanoparticles and B2 RAW 264.7 cells incubated with MNP-PEI-PEG nanoparticles. The cells were incubated at different concentrations as indicated over a 72-h incubation. Cell viability was determined using common assays including MTS assay (square), CellTiter-Blue assay (triangle), CellTiter-Glo assay (circle) and trypan blue counting (diamond). (n = 3 ± SE). Asterisk denotes significantly increased level of cell viability compared with trypan blue measurement (p < 0.05).
Mentions: An interesting phenomenon was observed when measuring the cell viability of both the SH-SY5Y (Figure 2A1, B1) and RAW 264.7 (Figure 2A2, B2) cells after incubation with the MNPs using MTS, CellTiter-Blue and CellTiter-Glo assays. At increased concentrations, the MNPs became attached to either the cell membrane or the bottom of the plate, appearing as a brown colour in the well (even after five washes with fresh culture media) (data not shown). The presence of these MNPs resulted in greater absorption readings in the MTS assay and thus significantly showed an overestimation of the cell viability (p < 0.05). This phenomenon could be explained by the adherence of the sticky polymers to the well surface or the positive amine groups being attracted to the negative charge of the cell membrane. After pegylation, the interference appeared to have been reduced; however, the value of cell viability that appeared was still larger compared to the visual inspection of viable cells under microscope (data not shown).

Bottom Line: Full understanding of the interactions between MNPs and mammalian cells is a critical issue for their applications.The Titer-Glo also experienced a small overestimation.To further understand the cytotoxic effect of the nanoparticles on these cells, reactive oxygen species production, lipid peroxidation and cell membrane integrity were investigated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Medical Science and Technology (IMSaT), Wilson House, 1 Wurzburg Loan, University of Dundee, Dundee, DD2 1FD, UK. l.y.wang@dundee.ac.uk.

ABSTRACT
Magnetic nanoparticles [MNPs] made from iron oxides have many applications in biomedicine. Full understanding of the interactions between MNPs and mammalian cells is a critical issue for their applications. In this study, MNPs were coated with poly(ethylenimine) [MNP-PEI] and poly(ethylene glycol) [MNP-PEI-PEG] to provide a subtle difference in their surface charge and their cytotoxicity which were analysed by three standard cell viability assays: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium [MTS], CellTiter-Blue and CellTiter-Glo (Promega, Southampton, UK) in SH-SY5Y and RAW 264.7 cells The data were validated by traditional trypan blue exclusion. In comparison to trypan blue manual counting, the MTS and Titer-Blue assays appeared to have consistently overestimated the viability. The Titer-Glo also experienced a small overestimation. We hypothesise that interactions were occurring between the assay systems and the nanoparticles, resulting in incorrect cell viability evaluation. To further understand the cytotoxic effect of the nanoparticles on these cells, reactive oxygen species production, lipid peroxidation and cell membrane integrity were investigated. After pegylation, the MNP-PEI-PEG possessed a lower positive surface charge and exhibited much improved biocompatibility compared to MNP-PEI, as demonstrated not only by a higher cell viability, but also by a markedly reduced oxidative stress and cell membrane damage. These findings highlight the importance of assay selection and of dissection of different cellular responses in in-vitro characterisation of nanostructures.

No MeSH data available.


Related in: MedlinePlus