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Dextran coated ultrafine superparamagnetic iron oxide nanoparticles: compatibility with common fluorometric and colorimetric dyes.

Griffiths SM, Singh N, Jenkins GJ, Williams PM, Orbaek AW, Barron AR, Wright CJ, Doak SH - Anal. Chem. (2011)

Bottom Line: In the present study, both forms of dUSPION caused an increase in MTS signal but a decrease in background signal from calcein and 3'-(p-aminophenyl) fluorescein (APF) and no effect on CyQUANT and EthD-1 fluorescence responses.Magnetite caused a decrease in fluorescence signal of DCFH, but it did not decrease fluorescence signal in the presence of the reactive oxygen species-inducer tert-butyl hydroperoxide (TBHP).This study emphasizes the importance of considering and controlling for possible interactions between NM and fluorometric/colorimetric dyes and, most importantly, the oxidation state of dUSPION that may confound their sensitivity and specificity.

View Article: PubMed Central - PubMed

Affiliation: Institute of Life Science, School of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP, Wales, UK.

ABSTRACT
Due to the unique physicochemical properties of nanomaterials (NM) and their unknown reactivity, the possibility of NM altering the optical properties of fluorometric/colorimetric probes that are used to measure their cyto- and genotoxicity may lead to inaccurate readings. This could have potential implications given that NM, such as ultrafine superparamagnetic iron oxide nanoparticles (USPION), are increasingly finding their use in nanomedicine and the absorbance/fluorescence based assays are used to assess their toxicity. This study looks at the potential of dextran-coated USPION (dUSPION) (maghemite and magnetite) to alter the background signal of common probes used for evaluating cytotoxicity (MTS, CyQUANT, Calcein, and EthD-1) and oxidative stress (DCFH-DA and APF). In the present study, both forms of dUSPION caused an increase in MTS signal but a decrease in background signal from calcein and 3'-(p-aminophenyl) fluorescein (APF) and no effect on CyQUANT and EthD-1 fluorescence responses. Magnetite caused a decrease in fluorescence signal of DCFH, but it did not decrease fluorescence signal in the presence of the reactive oxygen species-inducer tert-butyl hydroperoxide (TBHP). In contrast, maghemite caused an increase in fluorescence, which was substantially reduced in the presence of the antioxidant N-acetyl cysteine. This study emphasizes the importance of considering and controlling for possible interactions between NM and fluorometric/colorimetric dyes and, most importantly, the oxidation state of dUSPION that may confound their sensitivity and specificity.

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Effect of dUSPION exposure on APF fluorescence response in an acellular system, n = 3, significantly (*p < 0.05) different to untreated control.
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fig7: Effect of dUSPION exposure on APF fluorescence response in an acellular system, n = 3, significantly (*p < 0.05) different to untreated control.

Mentions: Similar to DCFH-DA, APF is a fluorometric probe used for the detection of oxidative species. APF is oxidized by free radicals to yield a highly fluorescent product which can be detected at 520 nm. Concentrations of maghemite and magnetite between 1 × 10–3 and 1 μg/mL did not have a significant effect on the resultant intensity of the APF fluorescence signal. However, 10 and 100 μg/mL maghemite and 100 μg/mL magnetite caused a significant decrease in background APF fluorescence signal (Figure 7), which could be due to adsorption onto the surface of the NPs, thus quenching the fluorescence response. This is in contrast to the results seen when using DCFH with maghemite, which reported increased oxidative stress at 1–100 μg/mL (Figure 6). This may be because APF is oxidized by fewer free radical species than DCFH as it is much more selective, only detecting the hydroxyl radical and peroxynitrite anion. Thus, APF may not be detecting the specific ROS produced by maghemite, which are possibly ROS products of Fenton-like reactions. The contrasting results obtained from using these two different oxidative stress assays again highlights the difficulties and complexity of testing the safety of NM.


Dextran coated ultrafine superparamagnetic iron oxide nanoparticles: compatibility with common fluorometric and colorimetric dyes.

Griffiths SM, Singh N, Jenkins GJ, Williams PM, Orbaek AW, Barron AR, Wright CJ, Doak SH - Anal. Chem. (2011)

Effect of dUSPION exposure on APF fluorescence response in an acellular system, n = 3, significantly (*p < 0.05) different to untreated control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3095146&req=5

fig7: Effect of dUSPION exposure on APF fluorescence response in an acellular system, n = 3, significantly (*p < 0.05) different to untreated control.
Mentions: Similar to DCFH-DA, APF is a fluorometric probe used for the detection of oxidative species. APF is oxidized by free radicals to yield a highly fluorescent product which can be detected at 520 nm. Concentrations of maghemite and magnetite between 1 × 10–3 and 1 μg/mL did not have a significant effect on the resultant intensity of the APF fluorescence signal. However, 10 and 100 μg/mL maghemite and 100 μg/mL magnetite caused a significant decrease in background APF fluorescence signal (Figure 7), which could be due to adsorption onto the surface of the NPs, thus quenching the fluorescence response. This is in contrast to the results seen when using DCFH with maghemite, which reported increased oxidative stress at 1–100 μg/mL (Figure 6). This may be because APF is oxidized by fewer free radical species than DCFH as it is much more selective, only detecting the hydroxyl radical and peroxynitrite anion. Thus, APF may not be detecting the specific ROS produced by maghemite, which are possibly ROS products of Fenton-like reactions. The contrasting results obtained from using these two different oxidative stress assays again highlights the difficulties and complexity of testing the safety of NM.

Bottom Line: In the present study, both forms of dUSPION caused an increase in MTS signal but a decrease in background signal from calcein and 3'-(p-aminophenyl) fluorescein (APF) and no effect on CyQUANT and EthD-1 fluorescence responses.Magnetite caused a decrease in fluorescence signal of DCFH, but it did not decrease fluorescence signal in the presence of the reactive oxygen species-inducer tert-butyl hydroperoxide (TBHP).This study emphasizes the importance of considering and controlling for possible interactions between NM and fluorometric/colorimetric dyes and, most importantly, the oxidation state of dUSPION that may confound their sensitivity and specificity.

View Article: PubMed Central - PubMed

Affiliation: Institute of Life Science, School of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP, Wales, UK.

ABSTRACT
Due to the unique physicochemical properties of nanomaterials (NM) and their unknown reactivity, the possibility of NM altering the optical properties of fluorometric/colorimetric probes that are used to measure their cyto- and genotoxicity may lead to inaccurate readings. This could have potential implications given that NM, such as ultrafine superparamagnetic iron oxide nanoparticles (USPION), are increasingly finding their use in nanomedicine and the absorbance/fluorescence based assays are used to assess their toxicity. This study looks at the potential of dextran-coated USPION (dUSPION) (maghemite and magnetite) to alter the background signal of common probes used for evaluating cytotoxicity (MTS, CyQUANT, Calcein, and EthD-1) and oxidative stress (DCFH-DA and APF). In the present study, both forms of dUSPION caused an increase in MTS signal but a decrease in background signal from calcein and 3'-(p-aminophenyl) fluorescein (APF) and no effect on CyQUANT and EthD-1 fluorescence responses. Magnetite caused a decrease in fluorescence signal of DCFH, but it did not decrease fluorescence signal in the presence of the reactive oxygen species-inducer tert-butyl hydroperoxide (TBHP). In contrast, maghemite caused an increase in fluorescence, which was substantially reduced in the presence of the antioxidant N-acetyl cysteine. This study emphasizes the importance of considering and controlling for possible interactions between NM and fluorometric/colorimetric dyes and, most importantly, the oxidation state of dUSPION that may confound their sensitivity and specificity.

Show MeSH
Related in: MedlinePlus