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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

Correlation between side scatter measurements and SPION-loads. HUVECs were cultured for 48 hours in medium containing 0–100 µgFe/mL SPIONs.Notes: (A) Correlation of the side scatter and the SPION concentration within the cell culture medium. (B) Relationship between the cellular SPION-load as measured by AAS and the normalized side scatter data delivered by flow cytometry. Results were acquired with SEONLA (left panel), SEONLA-BSA (middle panel), and Rienso® (right panel). Data are expressed as the mean ± standard error (n=3 with technical triplicates). R2 represents the coefficient of determination. y describes the mathematic relationship between side scatter and cellular SPION content.Abbreviations: SPION, superparamagnetic iron oxide nanoparticle; AAS, atomic adsorption spectroscopy; HUVECs, human umbilical vein endothelial cells; SEONLA, lauric acid-coated nanoparticles; SEONLA-BSA, lauric acid/albumin bovine serum hybrid-coated nanoparticles.
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f3-ijn-10-4185: Correlation between side scatter measurements and SPION-loads. HUVECs were cultured for 48 hours in medium containing 0–100 µgFe/mL SPIONs.Notes: (A) Correlation of the side scatter and the SPION concentration within the cell culture medium. (B) Relationship between the cellular SPION-load as measured by AAS and the normalized side scatter data delivered by flow cytometry. Results were acquired with SEONLA (left panel), SEONLA-BSA (middle panel), and Rienso® (right panel). Data are expressed as the mean ± standard error (n=3 with technical triplicates). R2 represents the coefficient of determination. y describes the mathematic relationship between side scatter and cellular SPION content.Abbreviations: SPION, superparamagnetic iron oxide nanoparticle; AAS, atomic adsorption spectroscopy; HUVECs, human umbilical vein endothelial cells; SEONLA, lauric acid-coated nanoparticles; SEONLA-BSA, lauric acid/albumin bovine serum hybrid-coated nanoparticles.

Mentions: In flow cytometry, cells can be morphologically characterized by forward scatter and side scatter, where the forward scatter is corresponding to cell “size” and the side scatter to “granularity” of the cell. As nanoparticles scatter light, it is possible to detect intracellular nanoparticles also by side scatter analysis in flow cytometry, if the side scatter increase of nanoparticle-treated cells compared with untreated cells surpasses the detection threshold (Table S1). Since the cellular size and granularity changes during cell death, it is necessary to exclude dying or dead cells with altered morphology from analysis of the side scatter for SPION uptake. Gating on phenotypically healthy cells, characterized by negative Annexin V and negative propidium iodide staining, a significant correlation was observed between the SPION amount in the cell culture medium and the side scatter of viable cells after 48 hours of incubation with SEONLA-BSA (R2>0.97). The SEONLA data demonstrated a similar correlation when narrowing the analysis to cells with a SPION-load below 100 µgFe/mL (R2>0.99; Figure 3A, left and middle panel). Cells incubated with Rienso® did not display a significant change in side scatter increase in response to the growing nanoparticle dose in the medium, confirming an extremely low cellular uptake or binding of these particles (Figure 3A, right panel), as compared with SEONLA-BSA and SEONLA par ticles. Figure S3 shows representative flow cytometric graphs of nanoparticle-treated cells, and highlights the principle of the side scatter increase after nanoparticle attachment/uptake by cells.


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)

Correlation between side scatter measurements and SPION-loads. HUVECs were cultured for 48 hours in medium containing 0–100 µgFe/mL SPIONs.Notes: (A) Correlation of the side scatter and the SPION concentration within the cell culture medium. (B) Relationship between the cellular SPION-load as measured by AAS and the normalized side scatter data delivered by flow cytometry. Results were acquired with SEONLA (left panel), SEONLA-BSA (middle panel), and Rienso® (right panel). Data are expressed as the mean ± standard error (n=3 with technical triplicates). R2 represents the coefficient of determination. y describes the mathematic relationship between side scatter and cellular SPION content.Abbreviations: SPION, superparamagnetic iron oxide nanoparticle; AAS, atomic adsorption spectroscopy; HUVECs, human umbilical vein endothelial cells; SEONLA, lauric acid-coated nanoparticles; SEONLA-BSA, lauric acid/albumin bovine serum hybrid-coated nanoparticles.
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Related In: Results  -  Collection

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f3-ijn-10-4185: Correlation between side scatter measurements and SPION-loads. HUVECs were cultured for 48 hours in medium containing 0–100 µgFe/mL SPIONs.Notes: (A) Correlation of the side scatter and the SPION concentration within the cell culture medium. (B) Relationship between the cellular SPION-load as measured by AAS and the normalized side scatter data delivered by flow cytometry. Results were acquired with SEONLA (left panel), SEONLA-BSA (middle panel), and Rienso® (right panel). Data are expressed as the mean ± standard error (n=3 with technical triplicates). R2 represents the coefficient of determination. y describes the mathematic relationship between side scatter and cellular SPION content.Abbreviations: SPION, superparamagnetic iron oxide nanoparticle; AAS, atomic adsorption spectroscopy; HUVECs, human umbilical vein endothelial cells; SEONLA, lauric acid-coated nanoparticles; SEONLA-BSA, lauric acid/albumin bovine serum hybrid-coated nanoparticles.
Mentions: In flow cytometry, cells can be morphologically characterized by forward scatter and side scatter, where the forward scatter is corresponding to cell “size” and the side scatter to “granularity” of the cell. As nanoparticles scatter light, it is possible to detect intracellular nanoparticles also by side scatter analysis in flow cytometry, if the side scatter increase of nanoparticle-treated cells compared with untreated cells surpasses the detection threshold (Table S1). Since the cellular size and granularity changes during cell death, it is necessary to exclude dying or dead cells with altered morphology from analysis of the side scatter for SPION uptake. Gating on phenotypically healthy cells, characterized by negative Annexin V and negative propidium iodide staining, a significant correlation was observed between the SPION amount in the cell culture medium and the side scatter of viable cells after 48 hours of incubation with SEONLA-BSA (R2>0.97). The SEONLA data demonstrated a similar correlation when narrowing the analysis to cells with a SPION-load below 100 µgFe/mL (R2>0.99; Figure 3A, left and middle panel). Cells incubated with Rienso® did not display a significant change in side scatter increase in response to the growing nanoparticle dose in the medium, confirming an extremely low cellular uptake or binding of these particles (Figure 3A, right panel), as compared with SEONLA-BSA and SEONLA par ticles. Figure S3 shows representative flow cytometric graphs of nanoparticle-treated cells, and highlights the principle of the side scatter increase after nanoparticle attachment/uptake by cells.

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