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In vitro cytotoxicity analysis of doxorubicin-loaded/superparamagnetic iron oxide colloidal nanoassemblies on MCF7 and NIH3T3 cell lines.

Tomankova K, Polakova K, Pizova K, Binder S, Havrdova M, Kolarova M, Kriegova E, Zapletalova J, Malina L, Horakova J, Malohlava J, Kolokithas-Ntoukas A, Bakandritsos A, Kolarova H, Zboril R - Int J Nanomedicine (2015)

Bottom Line: For proper analysis and understanding of cell behavior after administration of MagAlg-DOX compared with free DOX, a complex set of in vitro tests, including production of reactive oxygen species, comet assay, cell cycle determination, gene expression, and cellular uptake, were utilized.It was found that the cytotoxic effect of MagAlg-DOX system is delayed compared to free DOX in both cell lines.We discovered that nanoparticles can attenuate or even inhibit the effect of DOX, particularly in the tumor MCF7 cell line.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biophysics, Institute of Translation Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic.

ABSTRACT
One of the promising strategies for improvement of cancer treatment is based on magnetic drug delivery systems, thus avoiding side effects of standard chemotherapies. Superparamagnetic iron oxide (SPIO) nanoparticles have ideal properties to become a targeted magnetic drug delivery contrast probes, named theranostics. We worked with SPIO condensed colloidal nanocrystal clusters (MagAlg) prepared through a new soft biomineralization route in the presence of alginate as the polymeric shell and loaded with doxorubicin (DOX). The aim of this work was to study the in vitro cytotoxicity of these new MagAlg-DOX systems on mouse fibroblast and breast carcinoma cell lines. For proper analysis and understanding of cell behavior after administration of MagAlg-DOX compared with free DOX, a complex set of in vitro tests, including production of reactive oxygen species, comet assay, cell cycle determination, gene expression, and cellular uptake, were utilized. It was found that the cytotoxic effect of MagAlg-DOX system is delayed compared to free DOX in both cell lines. This was attributed to the different mechanism of internalization of DOX and MagAlg-DOX into the cells, together with the fact that the drug is strongly bound on the drug nanocarriers. We discovered that nanoparticles can attenuate or even inhibit the effect of DOX, particularly in the tumor MCF7 cell line. This is a first comprehensive study on the cytotoxic effect of DOX-loaded SPIO compared with free DOX on healthy and cancer cell lines, as well as on the induced changes in gene expression.

No MeSH data available.


Related in: MedlinePlus

Production of mitochondrial membrane potential changes in MCF7 and NIH3T3 cell lines.Notes: The ratio of median of green monomer and red aggregate expressed the amount created in dependence of concentration of 50 μM, 5 μM, and 0.5 μM of DOX or MagAlg–DOX nanocarrier. The higher values the higher probability of early stage of apoptosis. Data represent mean and standard error from three independent measurements. Significance was determined using ANOVA and Dunnet post hoc test.Abbreviations: DOX, doxorubicin; RFU, relative fluorescence unit.
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f3-ijn-10-949: Production of mitochondrial membrane potential changes in MCF7 and NIH3T3 cell lines.Notes: The ratio of median of green monomer and red aggregate expressed the amount created in dependence of concentration of 50 μM, 5 μM, and 0.5 μM of DOX or MagAlg–DOX nanocarrier. The higher values the higher probability of early stage of apoptosis. Data represent mean and standard error from three independent measurements. Significance was determined using ANOVA and Dunnet post hoc test.Abbreviations: DOX, doxorubicin; RFU, relative fluorescence unit.

Mentions: The effects of DOX and/or MagAlg–DOX nanocarriers on cell viability were determined using MTT assay. The absorbance measured, using this assay, reflects the total metabolic activity of a cell population and is therefore also an indirect measurement of cell proliferation. Although commonly used as a viability assay, the MTT assay more specifically represents a measure of mitochondrial function. The results from MTT viability tests are presented in Figure 2 and in the form of IC50 (Table 2). After 24 hours of incubation, a significant decrease in cell viability was found at all DOX concentrations in both groups (free-DOX and MagAlg–DOX) and in both the cell lines. Cell viability systematically decreased with increasing DOX concentration. However, low level (>80%) of cell viability can be seen only in DOX concentrations between 5 μM and 50 μM in both the cell lines of the free-DOX group. On the other hand, in MagAlg–DOX group, we found low cell viability only at the concentration of 50 μM in the MCF7 cell line. IC50 values show the higher cytotoxic effect of DOX (either free or bound to MagAlg) in the MCF7 cell line. IC50 values (Table 2) also manifest the significantly lower cytotoxicity of MagAlg–DOX (IC50 =18.245 μM of DOX) as compared to free-DOX (IC50 =1.2009 μM of DOX). Mitochondria are considered as one of the primary targets of DOX through mitochondria-mediated apoptosis associated with changes in mitochondrial functional parameters.17 The MMP change (Δψm) assay was used to evaluate these changes in cells 6 hours after treatment (Figure 3). The ratio of median formation of green monomer and red aggregate expresses the probability of apoptosis. Higher values reflect greater cell perturbation. However, statistical analysis showed no significant changes for individual concentrations. In the NIH3T3 cell line, higher values of mitochondrial membrane depolarization were observed in the early stage of the cell death process in concentrations 0.5 μM and 5 μM in the free-DOX group. In the MagAlg–DOX group, no significant changes were found in both the cell lines.


In vitro cytotoxicity analysis of doxorubicin-loaded/superparamagnetic iron oxide colloidal nanoassemblies on MCF7 and NIH3T3 cell lines.

Tomankova K, Polakova K, Pizova K, Binder S, Havrdova M, Kolarova M, Kriegova E, Zapletalova J, Malina L, Horakova J, Malohlava J, Kolokithas-Ntoukas A, Bakandritsos A, Kolarova H, Zboril R - Int J Nanomedicine (2015)

Production of mitochondrial membrane potential changes in MCF7 and NIH3T3 cell lines.Notes: The ratio of median of green monomer and red aggregate expressed the amount created in dependence of concentration of 50 μM, 5 μM, and 0.5 μM of DOX or MagAlg–DOX nanocarrier. The higher values the higher probability of early stage of apoptosis. Data represent mean and standard error from three independent measurements. Significance was determined using ANOVA and Dunnet post hoc test.Abbreviations: DOX, doxorubicin; RFU, relative fluorescence unit.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321606&req=5

f3-ijn-10-949: Production of mitochondrial membrane potential changes in MCF7 and NIH3T3 cell lines.Notes: The ratio of median of green monomer and red aggregate expressed the amount created in dependence of concentration of 50 μM, 5 μM, and 0.5 μM of DOX or MagAlg–DOX nanocarrier. The higher values the higher probability of early stage of apoptosis. Data represent mean and standard error from three independent measurements. Significance was determined using ANOVA and Dunnet post hoc test.Abbreviations: DOX, doxorubicin; RFU, relative fluorescence unit.
Mentions: The effects of DOX and/or MagAlg–DOX nanocarriers on cell viability were determined using MTT assay. The absorbance measured, using this assay, reflects the total metabolic activity of a cell population and is therefore also an indirect measurement of cell proliferation. Although commonly used as a viability assay, the MTT assay more specifically represents a measure of mitochondrial function. The results from MTT viability tests are presented in Figure 2 and in the form of IC50 (Table 2). After 24 hours of incubation, a significant decrease in cell viability was found at all DOX concentrations in both groups (free-DOX and MagAlg–DOX) and in both the cell lines. Cell viability systematically decreased with increasing DOX concentration. However, low level (>80%) of cell viability can be seen only in DOX concentrations between 5 μM and 50 μM in both the cell lines of the free-DOX group. On the other hand, in MagAlg–DOX group, we found low cell viability only at the concentration of 50 μM in the MCF7 cell line. IC50 values show the higher cytotoxic effect of DOX (either free or bound to MagAlg) in the MCF7 cell line. IC50 values (Table 2) also manifest the significantly lower cytotoxicity of MagAlg–DOX (IC50 =18.245 μM of DOX) as compared to free-DOX (IC50 =1.2009 μM of DOX). Mitochondria are considered as one of the primary targets of DOX through mitochondria-mediated apoptosis associated with changes in mitochondrial functional parameters.17 The MMP change (Δψm) assay was used to evaluate these changes in cells 6 hours after treatment (Figure 3). The ratio of median formation of green monomer and red aggregate expresses the probability of apoptosis. Higher values reflect greater cell perturbation. However, statistical analysis showed no significant changes for individual concentrations. In the NIH3T3 cell line, higher values of mitochondrial membrane depolarization were observed in the early stage of the cell death process in concentrations 0.5 μM and 5 μM in the free-DOX group. In the MagAlg–DOX group, no significant changes were found in both the cell lines.

Bottom Line: For proper analysis and understanding of cell behavior after administration of MagAlg-DOX compared with free DOX, a complex set of in vitro tests, including production of reactive oxygen species, comet assay, cell cycle determination, gene expression, and cellular uptake, were utilized.It was found that the cytotoxic effect of MagAlg-DOX system is delayed compared to free DOX in both cell lines.We discovered that nanoparticles can attenuate or even inhibit the effect of DOX, particularly in the tumor MCF7 cell line.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biophysics, Institute of Translation Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic.

ABSTRACT
One of the promising strategies for improvement of cancer treatment is based on magnetic drug delivery systems, thus avoiding side effects of standard chemotherapies. Superparamagnetic iron oxide (SPIO) nanoparticles have ideal properties to become a targeted magnetic drug delivery contrast probes, named theranostics. We worked with SPIO condensed colloidal nanocrystal clusters (MagAlg) prepared through a new soft biomineralization route in the presence of alginate as the polymeric shell and loaded with doxorubicin (DOX). The aim of this work was to study the in vitro cytotoxicity of these new MagAlg-DOX systems on mouse fibroblast and breast carcinoma cell lines. For proper analysis and understanding of cell behavior after administration of MagAlg-DOX compared with free DOX, a complex set of in vitro tests, including production of reactive oxygen species, comet assay, cell cycle determination, gene expression, and cellular uptake, were utilized. It was found that the cytotoxic effect of MagAlg-DOX system is delayed compared to free DOX in both cell lines. This was attributed to the different mechanism of internalization of DOX and MagAlg-DOX into the cells, together with the fact that the drug is strongly bound on the drug nanocarriers. We discovered that nanoparticles can attenuate or even inhibit the effect of DOX, particularly in the tumor MCF7 cell line. This is a first comprehensive study on the cytotoxic effect of DOX-loaded SPIO compared with free DOX on healthy and cancer cell lines, as well as on the induced changes in gene expression.

No MeSH data available.


Related in: MedlinePlus