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

TEM images show cellular uptake of magnetic nanoparticles. A1 MNP incubated with SH-SY5Y cells, A2 MNP incubated with RAW 264.7 cells, B1 MNP-PEI incubated with SH-SY5Y cells, B2 MNP-PEI incubated with RAW 264.7 cells, C1 MNP-PEI-PEG incubated with SH-SY5Y cells and C2 MNP-PEI-PEG incubated with RAW 264.7 cells. Samples were incubated with cells at 6.25 μg mL-1 for 24 h, and internalisation of nanoparticles was analysed by TEM as described in the 'Methods' section.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: TEM images show cellular uptake of magnetic nanoparticles. A1 MNP incubated with SH-SY5Y cells, A2 MNP incubated with RAW 264.7 cells, B1 MNP-PEI incubated with SH-SY5Y cells, B2 MNP-PEI incubated with RAW 264.7 cells, C1 MNP-PEI-PEG incubated with SH-SY5Y cells and C2 MNP-PEI-PEG incubated with RAW 264.7 cells. Samples were incubated with cells at 6.25 μg mL-1 for 24 h, and internalisation of nanoparticles was analysed by TEM as described in the 'Methods' section.

Mentions: The TEM micrographs were consistent with the ICP data, for the SH-SY5Y cells increased numbers of intracellular nanoparticles were evident upon coating the particles with PEI and PEG (Figure 1A1, B1, C1). The RAW 264.7 images (Figure 1A2, B2, C2) were also in good agreement with the ICP measurement whereby PEI and PEG coating had less effect on the total cellular uptake of the nanoparticles.


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)

TEM images show cellular uptake of magnetic nanoparticles. A1 MNP incubated with SH-SY5Y cells, A2 MNP incubated with RAW 264.7 cells, B1 MNP-PEI incubated with SH-SY5Y cells, B2 MNP-PEI incubated with RAW 264.7 cells, C1 MNP-PEI-PEG incubated with SH-SY5Y cells and C2 MNP-PEI-PEG incubated with RAW 264.7 cells. Samples were incubated with cells at 6.25 μg mL-1 for 24 h, and internalisation of nanoparticles was analysed by TEM as described in the 'Methods' section.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: TEM images show cellular uptake of magnetic nanoparticles. A1 MNP incubated with SH-SY5Y cells, A2 MNP incubated with RAW 264.7 cells, B1 MNP-PEI incubated with SH-SY5Y cells, B2 MNP-PEI incubated with RAW 264.7 cells, C1 MNP-PEI-PEG incubated with SH-SY5Y cells and C2 MNP-PEI-PEG incubated with RAW 264.7 cells. Samples were incubated with cells at 6.25 μg mL-1 for 24 h, and internalisation of nanoparticles was analysed by TEM as described in the 'Methods' section.
Mentions: The TEM micrographs were consistent with the ICP data, for the SH-SY5Y cells increased numbers of intracellular nanoparticles were evident upon coating the particles with PEI and PEG (Figure 1A1, B1, C1). The RAW 264.7 images (Figure 1A2, B2, C2) were also in good agreement with the ICP measurement whereby PEI and PEG coating had less effect on the total cellular uptake of the nanoparticles.

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