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Flow cytometry for intracellular SPION quantification: specificity and sensitivity in comparison with spectroscopic methods.

Friedrich RP, Janko C, Poettler M, Tripal P, Zaloga J, Cicha I, Dürr S, Nowak J, Odenbach S, Slabu I, Liebl M, Trahms L, Stapf M, Hilger I, Lyer S, Alexiou C - Int J Nanomedicine (2015)

Bottom Line: In the present study, we compared three different SPION quantification methods (ultraviolet spectrophotometry, magnetic particle spectroscopy, atomic adsorption spectroscopy) and discussed the shortcomings and advantages of each method.Our data also demonstrate that internalization of iron oxide nanoparticles in human umbilical vein endothelial cells is strongly dependent to the SPION type and results in a dose-dependent increase of toxicity.In summary, our data show that flow cytometry analysis can be used for estimation of uptake of SPIONs by mammalian cells and provides a fast tool for scientists to evaluate the safety of nanoparticle products.

View Article: PubMed Central - PubMed

Affiliation: Department of Otorhinolaryngology, Head and Neck Surgery, Section of Experimental Oncology and Nanomedicine, University hospital Erlangen, Erlangen, Germany.

ABSTRACT
Due to their special physicochemical properties, iron nanoparticles offer new promising possibilities for biomedical applications. For bench to bedside translation of super-paramagnetic iron oxide nanoparticles (SPIONs), safety issues have to be comprehensively clarified. To understand concentration-dependent nanoparticle-mediated toxicity, the exact quantification of intracellular SPIONs by reliable methods is of great importance. In the present study, we compared three different SPION quantification methods (ultraviolet spectrophotometry, magnetic particle spectroscopy, atomic adsorption spectroscopy) and discussed the shortcomings and advantages of each method. Moreover, we used those results to evaluate the possibility to use flow cytometric technique to determine the cellular SPION content. For this purpose, we correlated the side scatter data received from flow cytometry with the actual cellular SPION amount. We showed that flow cytometry provides a rapid and reliable method to assess the cellular SPION content. Our data also demonstrate that internalization of iron oxide nanoparticles in human umbilical vein endothelial cells is strongly dependent to the SPION type and results in a dose-dependent increase of toxicity. Thus, treatment with lauric acid-coated SPIONs (SEON(LA)) resulted in a significant increase in the intensity of side scatter and toxicity, whereas SEON(LA) with an additional protein corona formed by bovine serum albumin (SEON(LA-BSA)) and commercially available Rienso(®) particles showed only a minimal increase in both side scatter intensity and cellular toxicity. The increase in side scatter was in accordance with the measurements for SPION content by the atomic adsorption spectroscopy reference method. In summary, our data show that flow cytometry analysis can be used for estimation of uptake of SPIONs by mammalian cells and provides a fast tool for scientists to evaluate the safety of nanoparticle products.

No MeSH data available.


Related in: MedlinePlus

Suitability of UVS, MPS, and AAS techniques for measurement of SPION concentration in cell lysates.Notes: (A) UVS measurements at 370 nm, (B) MPS measurements, and (C) AAS measurements with increasing SPION concentration in cell lysates. Data are presented as the mean ± standard deviation (n=3 with triplicates). R2 represents the coefficient of determination.Abbreviations: au, absorbance units; SPION, superparamagnetic iron oxide nanoparticle; UVS, ultraviolet spectrophotometry; MPS, magnetic particle spectroscopy; AAS, atomic adsorption spectroscopy; SEONLA, lauric acid-coated nanoparticles; SEONLA-BSA, lauric acid/albumin bovine serum hybrid-coated nanoparticles; OD370, optical density at 370 nm.
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f1-ijn-10-4185: Suitability of UVS, MPS, and AAS techniques for measurement of SPION concentration in cell lysates.Notes: (A) UVS measurements at 370 nm, (B) MPS measurements, and (C) AAS measurements with increasing SPION concentration in cell lysates. Data are presented as the mean ± standard deviation (n=3 with triplicates). R2 represents the coefficient of determination.Abbreviations: au, absorbance units; SPION, superparamagnetic iron oxide nanoparticle; UVS, ultraviolet spectrophotometry; MPS, magnetic particle spectroscopy; AAS, atomic adsorption spectroscopy; SEONLA, lauric acid-coated nanoparticles; SEONLA-BSA, lauric acid/albumin bovine serum hybrid-coated nanoparticles; OD370, optical density at 370 nm.

Mentions: To quantify the cellular SPION content, we modified a photometric technique recently published by Dadashzadeh et al.22 Using this method, the absorption maximum for SPIONs should be observable at 370 nm. To ensure that this also applies to the SPIONs used in this study, we prepared SPION dilutions of 30 µgFe/mL in 10% sodium dodecyl sulfate and monitored the corresponding absorption spectra between 200 nm and 900 nm with a stepwise increase of 2 nm. Beside an obvious absorption maximum at ~222 nm, a second absorption peak is observable at a wavelength of roughlŷ370 nm (Figure S2A). To test this method for reproducibility and for medium-throughput suitability in 96-well format, we measured the absorption of 50 µL dilutions of SPIONs (0–50 µgFe/mL) in 10% sodium dodecyl sulfate at 370 nm and found a direct correlation between absorption and concentration of the particles (Figure S2B–D). Another very important aspect for quantification of SPIONs within cell lysates by a photometric approach is that the absorption of cellular components should not interfere with the absorption peak of SPIONs at 370 nm. Therefore, the reliability of the described method was verified by adding different SPION concentrations (0–50 µgFe/mL iron) into HUVEC lysates (Figure 1A). Even in these settings, an almost perfect correlation was observed between the measured absorption and quantity of added SPIONs, demonstrating the high degree of consistency for determination of the SPION concentration in cell lysates with this method.


Flow cytometry for intracellular SPION quantification: specificity and sensitivity in comparison with spectroscopic methods.

Friedrich RP, Janko C, Poettler M, Tripal P, Zaloga J, Cicha I, Dürr S, Nowak J, Odenbach S, Slabu I, Liebl M, Trahms L, Stapf M, Hilger I, Lyer S, Alexiou C - Int J Nanomedicine (2015)

Suitability of UVS, MPS, and AAS techniques for measurement of SPION concentration in cell lysates.Notes: (A) UVS measurements at 370 nm, (B) MPS measurements, and (C) AAS measurements with increasing SPION concentration in cell lysates. Data are presented as the mean ± standard deviation (n=3 with triplicates). R2 represents the coefficient of determination.Abbreviations: au, absorbance units; SPION, superparamagnetic iron oxide nanoparticle; UVS, ultraviolet spectrophotometry; MPS, magnetic particle spectroscopy; AAS, atomic adsorption spectroscopy; SEONLA, lauric acid-coated nanoparticles; SEONLA-BSA, lauric acid/albumin bovine serum hybrid-coated nanoparticles; OD370, optical density at 370 nm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492632&req=5

f1-ijn-10-4185: Suitability of UVS, MPS, and AAS techniques for measurement of SPION concentration in cell lysates.Notes: (A) UVS measurements at 370 nm, (B) MPS measurements, and (C) AAS measurements with increasing SPION concentration in cell lysates. Data are presented as the mean ± standard deviation (n=3 with triplicates). R2 represents the coefficient of determination.Abbreviations: au, absorbance units; SPION, superparamagnetic iron oxide nanoparticle; UVS, ultraviolet spectrophotometry; MPS, magnetic particle spectroscopy; AAS, atomic adsorption spectroscopy; SEONLA, lauric acid-coated nanoparticles; SEONLA-BSA, lauric acid/albumin bovine serum hybrid-coated nanoparticles; OD370, optical density at 370 nm.
Mentions: To quantify the cellular SPION content, we modified a photometric technique recently published by Dadashzadeh et al.22 Using this method, the absorption maximum for SPIONs should be observable at 370 nm. To ensure that this also applies to the SPIONs used in this study, we prepared SPION dilutions of 30 µgFe/mL in 10% sodium dodecyl sulfate and monitored the corresponding absorption spectra between 200 nm and 900 nm with a stepwise increase of 2 nm. Beside an obvious absorption maximum at ~222 nm, a second absorption peak is observable at a wavelength of roughlŷ370 nm (Figure S2A). To test this method for reproducibility and for medium-throughput suitability in 96-well format, we measured the absorption of 50 µL dilutions of SPIONs (0–50 µgFe/mL) in 10% sodium dodecyl sulfate at 370 nm and found a direct correlation between absorption and concentration of the particles (Figure S2B–D). Another very important aspect for quantification of SPIONs within cell lysates by a photometric approach is that the absorption of cellular components should not interfere with the absorption peak of SPIONs at 370 nm. Therefore, the reliability of the described method was verified by adding different SPION concentrations (0–50 µgFe/mL iron) into HUVEC lysates (Figure 1A). Even in these settings, an almost perfect correlation was observed between the measured absorption and quantity of added SPIONs, demonstrating the high degree of consistency for determination of the SPION concentration in cell lysates with this method.

Bottom Line: In the present study, we compared three different SPION quantification methods (ultraviolet spectrophotometry, magnetic particle spectroscopy, atomic adsorption spectroscopy) and discussed the shortcomings and advantages of each method.Our data also demonstrate that internalization of iron oxide nanoparticles in human umbilical vein endothelial cells is strongly dependent to the SPION type and results in a dose-dependent increase of toxicity.In summary, our data show that flow cytometry analysis can be used for estimation of uptake of SPIONs by mammalian cells and provides a fast tool for scientists to evaluate the safety of nanoparticle products.

View Article: PubMed Central - PubMed

Affiliation: Department of Otorhinolaryngology, Head and Neck Surgery, Section of Experimental Oncology and Nanomedicine, University hospital Erlangen, Erlangen, Germany.

ABSTRACT
Due to their special physicochemical properties, iron nanoparticles offer new promising possibilities for biomedical applications. For bench to bedside translation of super-paramagnetic iron oxide nanoparticles (SPIONs), safety issues have to be comprehensively clarified. To understand concentration-dependent nanoparticle-mediated toxicity, the exact quantification of intracellular SPIONs by reliable methods is of great importance. In the present study, we compared three different SPION quantification methods (ultraviolet spectrophotometry, magnetic particle spectroscopy, atomic adsorption spectroscopy) and discussed the shortcomings and advantages of each method. Moreover, we used those results to evaluate the possibility to use flow cytometric technique to determine the cellular SPION content. For this purpose, we correlated the side scatter data received from flow cytometry with the actual cellular SPION amount. We showed that flow cytometry provides a rapid and reliable method to assess the cellular SPION content. Our data also demonstrate that internalization of iron oxide nanoparticles in human umbilical vein endothelial cells is strongly dependent to the SPION type and results in a dose-dependent increase of toxicity. Thus, treatment with lauric acid-coated SPIONs (SEON(LA)) resulted in a significant increase in the intensity of side scatter and toxicity, whereas SEON(LA) with an additional protein corona formed by bovine serum albumin (SEON(LA-BSA)) and commercially available Rienso(®) particles showed only a minimal increase in both side scatter intensity and cellular toxicity. The increase in side scatter was in accordance with the measurements for SPION content by the atomic adsorption spectroscopy reference method. In summary, our data show that flow cytometry analysis can be used for estimation of uptake of SPIONs by mammalian cells and provides a fast tool for scientists to evaluate the safety of nanoparticle products.

No MeSH data available.


Related in: MedlinePlus