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

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.

Keywords: DOX/SPIO nanocarriers; doxorubicin; in vitro cytotoxicity; superparamagnetic iron oxide nanoparticles.

Figure 1

Kinetic production of reactive oxygen species in concentration of 50 μM, 5 μM, and 0.5 μM of DOX or MagAlg–DOX nanocarrier in MCF7 and NIH3T3 cell lines. Notes: The linear regression of ROS rate expressed the ROS amount created at each minutes. Data represent mean and standard error from three independent measurements. Positive (*) significance were determined using ANOVA and Dunnet post hoc test. Abbreviations: DOX, doxorubicin; ROS, reactive oxygen species; RFU, relative fluorescence unit.

Figure 2

The effect of DOX or MagAlg–DOX nanocarrier in concentration of 50 μM, 5 μM, and 0.5 μM on cell line viability of MCF7 and NIH3T3 cell lines. Notes: Data represent mean and standard error from three independent measurements. Negative (•) significance were determined using ANOVA and Dunnet post hoc test. Abbreviation: DOX, doxorubicin.

Figure 3

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.

Figure 4

Comet length (A and B) and percentage DNA in tail (C and D) determined by comet assay in concentration of 50 μM, 5 μM, and 0.5 μM of DOX and MagAlg–DOX nanocarrier on MCF7 and NIH3T3 cell lines. Notes: Data represent mean and standard error from three independent measurements. Positive (*) and negative (•) significance were determined using Mann–Whitney U-test with Bonferroni correction for multiple comparisons significance and Kruskal–Wallis test. Abbreviation: DOX, doxorubicin.

Figure 5

The influence of DOX and MagAlg–DOX nanocarrier in concentration of 5 μM, 0.5 μM, and 0 μM on cell cycle of MCF7 and NIH3T3 cell lines. Notes: Data represent mean and standard error from three independent measurements. Positive (*) and negative (•) significance were determined using χ² test with Bonferroni correction for multiple comparisons, and the method of adjusted residuals. Abbreviation: DOX, doxorubicin.

Figure 6

The influence of DOX and MagAlg–DOX nanocarrier in concentration of 5 μM, 0.5 μM, and 0 μM on phosphorylation of histone H3 in MCF7 and NIH3T3 cell lines. Notes: Data represent mean and standard error from three independent measurements. Negative (•) significance were determined using Fisher’s exact test with Bonferroni correction for multiple comparisons. Abbreviation: DOX, doxorubicin.

Figure 7

Fold change of C-MYC (A and B) and C-FOS (C and D) expression on MCF7 cells (left graphs) and NIH3T3 cells (right graphs) in the course of 0 μM, 0.5 μM, and 5 μM in DOX concentration and with effect of MagAlg–DOX nanocarrier. Notes: Data represent mean and standard error from three independent measurements. Positive (*) and negative (•) significance were determined using ANOVA and Dunnet post hoc test. Abbreviation: DOX, doxorubicin.

Figure 8

Atomic force microscopy of SPIO particles (A). Scan rate 0.3 Hz, scan size 1 μm², the height of NPs is 0 (dark part)–34 (light part) nm in z axis. Image resolution is 256 pixels. TEM images (B) of densely packed condensed clusters of SPIO nanoparticles. Abbreviations: SPIO, superparamagnetic iron oxide; TEM, transmission electron microscopy; NPs, nanoparticles.

Figure 9

In vitro fluorescent and Merge images of subcellular DOX distribution in: (A) MCF7 and NIH3T3 cells after 1 hour of incubation with DOX and MagAlg-DOX respectively, (B) in MCF7 and NIH3T3 cells after 24 hours incubation with free DOX and (C) in MCF7 (left) and NIH3T3 (right) cells after 6 hours of incubation with DOX and MagAlg-DOX respectively. The concentration of DOX was in all samples 50 μM. Abbreviations: DOX, doxorubicin; h, hours