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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

Association of cellular uptake with toxicity of SPIONs. HUVECs were incubated for 48 hours with different amounts of SPIONs and cytotoxicity was analyzed by flow cytometry.Notes: (A) Quantification of the absolute amount of total cell numbers and viable cells using MUSE. (B) Cell viability determined by Annexin V/propidium iodide staining. Percentages of necrotic (propidium iodide-positive), apoptotic (Annexin V-positive, propidium iodide-negative) and viable cells (Annexin V-negative, propidium iodide-negative) are shown. (C) Membrane potential analyzed by 1,1′,3,3,3′,3′-hexamethyl-indodicarbocyanine iodide [DiIC1(5)] staining. Graphs show cells with intact (DiI+) and depolarized (DiI−) membrane potential. (D) Cell cycle analysis by propidium iodide-Triton X staining. The cell status is expressed as the amount of apoptotic (degraded DNA, subG1), diploid (G1-phase) and double diploid (synthesis/G2 phase) cells. Data are presented as the mean ± standard error (n=3 with sample triplicates). The results were normalized to total cells (A) or to untreated control cells (B–D), set to 100%.Abbreviations: SPION, superparamagnetic iron oxide nanoparticle; HUVECs, human umbilical vein endothelial cells; SEONLA, lauric acid-coated nanoparticles; SEONLA-BSA, lauric acid/albumin bovine serum hybrid-coated nanoparticles; ctrl, control.
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f4-ijn-10-4185: Association of cellular uptake with toxicity of SPIONs. HUVECs were incubated for 48 hours with different amounts of SPIONs and cytotoxicity was analyzed by flow cytometry.Notes: (A) Quantification of the absolute amount of total cell numbers and viable cells using MUSE. (B) Cell viability determined by Annexin V/propidium iodide staining. Percentages of necrotic (propidium iodide-positive), apoptotic (Annexin V-positive, propidium iodide-negative) and viable cells (Annexin V-negative, propidium iodide-negative) are shown. (C) Membrane potential analyzed by 1,1′,3,3,3′,3′-hexamethyl-indodicarbocyanine iodide [DiIC1(5)] staining. Graphs show cells with intact (DiI+) and depolarized (DiI−) membrane potential. (D) Cell cycle analysis by propidium iodide-Triton X staining. The cell status is expressed as the amount of apoptotic (degraded DNA, subG1), diploid (G1-phase) and double diploid (synthesis/G2 phase) cells. Data are presented as the mean ± standard error (n=3 with sample triplicates). The results were normalized to total cells (A) or to untreated control cells (B–D), set to 100%.Abbreviations: SPION, superparamagnetic iron oxide nanoparticle; HUVECs, human umbilical vein endothelial cells; SEONLA, lauric acid-coated nanoparticles; SEONLA-BSA, lauric acid/albumin bovine serum hybrid-coated nanoparticles; ctrl, control.

Mentions: Flow cytometry is a rapid and comprehensive method for analysis of multiple toxicity parameters. The absolute amount of viable cells after treatment is a direct indicator of the toxic effect of SPIONs on cells. In our study, this parameter was routinely collected prior to determination of the cellular SPION-load and other toxicity parameters using the MUSE Cell Analyzer (Figure 4A). Interestingly, incubation of cells with SEONLA for 48 hours resulted in dramatic inhibition of proliferation, whereas this effect was much less pronounced upon increasing SEONLA-BSA treatment and absent with Rienso® particles.


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)

Association of cellular uptake with toxicity of SPIONs. HUVECs were incubated for 48 hours with different amounts of SPIONs and cytotoxicity was analyzed by flow cytometry.Notes: (A) Quantification of the absolute amount of total cell numbers and viable cells using MUSE. (B) Cell viability determined by Annexin V/propidium iodide staining. Percentages of necrotic (propidium iodide-positive), apoptotic (Annexin V-positive, propidium iodide-negative) and viable cells (Annexin V-negative, propidium iodide-negative) are shown. (C) Membrane potential analyzed by 1,1′,3,3,3′,3′-hexamethyl-indodicarbocyanine iodide [DiIC1(5)] staining. Graphs show cells with intact (DiI+) and depolarized (DiI−) membrane potential. (D) Cell cycle analysis by propidium iodide-Triton X staining. The cell status is expressed as the amount of apoptotic (degraded DNA, subG1), diploid (G1-phase) and double diploid (synthesis/G2 phase) cells. Data are presented as the mean ± standard error (n=3 with sample triplicates). The results were normalized to total cells (A) or to untreated control cells (B–D), set to 100%.Abbreviations: SPION, superparamagnetic iron oxide nanoparticle; HUVECs, human umbilical vein endothelial cells; SEONLA, lauric acid-coated nanoparticles; SEONLA-BSA, lauric acid/albumin bovine serum hybrid-coated nanoparticles; ctrl, control.
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Related In: Results  -  Collection

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f4-ijn-10-4185: Association of cellular uptake with toxicity of SPIONs. HUVECs were incubated for 48 hours with different amounts of SPIONs and cytotoxicity was analyzed by flow cytometry.Notes: (A) Quantification of the absolute amount of total cell numbers and viable cells using MUSE. (B) Cell viability determined by Annexin V/propidium iodide staining. Percentages of necrotic (propidium iodide-positive), apoptotic (Annexin V-positive, propidium iodide-negative) and viable cells (Annexin V-negative, propidium iodide-negative) are shown. (C) Membrane potential analyzed by 1,1′,3,3,3′,3′-hexamethyl-indodicarbocyanine iodide [DiIC1(5)] staining. Graphs show cells with intact (DiI+) and depolarized (DiI−) membrane potential. (D) Cell cycle analysis by propidium iodide-Triton X staining. The cell status is expressed as the amount of apoptotic (degraded DNA, subG1), diploid (G1-phase) and double diploid (synthesis/G2 phase) cells. Data are presented as the mean ± standard error (n=3 with sample triplicates). The results were normalized to total cells (A) or to untreated control cells (B–D), set to 100%.Abbreviations: SPION, superparamagnetic iron oxide nanoparticle; HUVECs, human umbilical vein endothelial cells; SEONLA, lauric acid-coated nanoparticles; SEONLA-BSA, lauric acid/albumin bovine serum hybrid-coated nanoparticles; ctrl, control.
Mentions: Flow cytometry is a rapid and comprehensive method for analysis of multiple toxicity parameters. The absolute amount of viable cells after treatment is a direct indicator of the toxic effect of SPIONs on cells. In our study, this parameter was routinely collected prior to determination of the cellular SPION-load and other toxicity parameters using the MUSE Cell Analyzer (Figure 4A). Interestingly, incubation of cells with SEONLA for 48 hours resulted in dramatic inhibition of proliferation, whereas this effect was much less pronounced upon increasing SEONLA-BSA treatment and absent with Rienso® particles.

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