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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.


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Concentration measurements of cellular SPIONs.Notes: (A–C) HUVECs were cultured for 48 hours in medium containing 0–100 µgFe/mL SPIONs. The measured cellular SPION concentration is given in pgFe/cell and µgFe/mL cell lysate and is correlated to the SPION concentration in medium indicated as µgFe/mL medium and µgFe/cm2 cell culture plate surface. Cellular SPION concentrations were measured with (A) UVS, (B) MPS, and (C) AAS. The columns present data 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.Abbreviations: 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; HUVECs, human umbilical vein endothelial cells.
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f2-ijn-10-4185: Concentration measurements of cellular SPIONs.Notes: (A–C) HUVECs were cultured for 48 hours in medium containing 0–100 µgFe/mL SPIONs. The measured cellular SPION concentration is given in pgFe/cell and µgFe/mL cell lysate and is correlated to the SPION concentration in medium indicated as µgFe/mL medium and µgFe/cm2 cell culture plate surface. Cellular SPION concentrations were measured with (A) UVS, (B) MPS, and (C) AAS. The columns present data 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.Abbreviations: 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; HUVECs, human umbilical vein endothelial cells.

Mentions: The amount of cellular SPION uptake was previously shown to depend on the concentration of SPIONs within the cell culture medium.24 To evaluate the suitability of each method to quantify cellular or cell-associated magnetic particles, as well as to correlate those findings with the flow cytometry analysis of the side scatter, HUVECs were treated with 0–100 µgFe/mL SPIONs for 48 hours (Figure 2A–C). Within the tested concentration range, the cellular uptake of SEONLA and SEONLA-BSA by HUVECs directly correlated with the SPION concentration in the cell culture medium (R2>0.96, R2>0.98, and R2>0.97 for the UVS, MPS, and AAS measurements, respectively, Figure 2, left and middle panel). In contrast, cell-associated Rienso® particles were hardly detectable by any of the methods (Figure 2, right panel). Even at the highest concentration, Rienso® particles were detected only with the AAS method, suggesting a very low uptake or binding of Rienso® to HUVECs.


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)

Concentration measurements of cellular SPIONs.Notes: (A–C) HUVECs were cultured for 48 hours in medium containing 0–100 µgFe/mL SPIONs. The measured cellular SPION concentration is given in pgFe/cell and µgFe/mL cell lysate and is correlated to the SPION concentration in medium indicated as µgFe/mL medium and µgFe/cm2 cell culture plate surface. Cellular SPION concentrations were measured with (A) UVS, (B) MPS, and (C) AAS. The columns present data 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.Abbreviations: 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; HUVECs, human umbilical vein endothelial cells.
© Copyright Policy
Related In: Results  -  Collection

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

f2-ijn-10-4185: Concentration measurements of cellular SPIONs.Notes: (A–C) HUVECs were cultured for 48 hours in medium containing 0–100 µgFe/mL SPIONs. The measured cellular SPION concentration is given in pgFe/cell and µgFe/mL cell lysate and is correlated to the SPION concentration in medium indicated as µgFe/mL medium and µgFe/cm2 cell culture plate surface. Cellular SPION concentrations were measured with (A) UVS, (B) MPS, and (C) AAS. The columns present data 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.Abbreviations: 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; HUVECs, human umbilical vein endothelial cells.
Mentions: The amount of cellular SPION uptake was previously shown to depend on the concentration of SPIONs within the cell culture medium.24 To evaluate the suitability of each method to quantify cellular or cell-associated magnetic particles, as well as to correlate those findings with the flow cytometry analysis of the side scatter, HUVECs were treated with 0–100 µgFe/mL SPIONs for 48 hours (Figure 2A–C). Within the tested concentration range, the cellular uptake of SEONLA and SEONLA-BSA by HUVECs directly correlated with the SPION concentration in the cell culture medium (R2>0.96, R2>0.98, and R2>0.97 for the UVS, MPS, and AAS measurements, respectively, Figure 2, left and middle panel). In contrast, cell-associated Rienso® particles were hardly detectable by any of the methods (Figure 2, right panel). Even at the highest concentration, Rienso® particles were detected only with the AAS method, suggesting a very low uptake or binding of Rienso® to HUVECs.

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