Shape-dependent cellular uptake of iron oxide nanorods: mechanisms of endocytosis and implications on cell labeling and cellular delivery

Abstract

The effects of nanoparticle morphology, especially size and shape, on their interactions with cells are of great interest in understanding the fate of nanoparticles in biological systems and designing them for biomedical applications. While size and shape-dependent cell behavior, endocytosis mechanism, and subcellular distribution of nanoparticles have been investigated extensively with gold and other nanoparticles, studies on iron oxide nanoparticles (IONP), one of the most promising and well-thought-of nanomaterials in biomedical applications, were limited. In this study, we synthesized oligosaccharide-coated water-soluble iron oxide nanorods (IONR) with different core sizes (nm) and different aspect ratios (i.e., length/width), such as IONR_(L) at 140/6 nm and IONR_(S) at 50/7 nm as well as spherical IONP (20 nm). We investigated how their sizes and shapes affect uptake mechanisms, localization, and cell viability in different cell lines. The results of transmission electron microscopy (TEM) and confocal fluorescence microscopic imaging confirmed the internalization of these nanoparticles in different types of cells and subsequent accumulation in the subcellular compartments, such as the endosomes, and into the cytosol. Specifically, IONR_(L) exhibited the highest cellular uptake compared to IONR_(S) and spherical IONP, 1.36-fold and 1.17-fold higher than that of spherical IONP in macrophages and pediatric brain tumor medulloblastoma cells, respectively. To examine the cellular uptake mechanisms preferred by the different IONR and IONP, we used different endocytosis inhibitors to block specific cellular internalization pathways when cells were treated with different nanoparticles. The results from these blocking experiments showed that IONR_(L) enter macrophages and normal kidney cells through clathrin-mediated, dynamin-dependent, and macropinocytosis/phagocytosis pathways, while they are internalized in cancer cells primarily via clathrin/caveolae-mediated and phagocytosis mechanisms. Overall, our findings provide new insights into further development of magnetic IONR-based imaging probes and drug delivery systems for biomedical applications.

Fig. 1. Transmission electron microscopy (TEM) images of oligosaccharide coated (a) IONR_(L), (b) IONR_(S), and (c) IONP and dynamic light scattering (DLS) measured (d) size distribution and (e) zeta potential of oligosaccharide coated IONR_(L), IONR_(S), and IONP. The XRD spectrum of oleylamine coated IONR_(L) (f) with red lines indicating the XRD reference pattern of magnetite (m).

Fig. 2. Comparison of the levels of cellular uptake of different IONR and IONP by (a) Raw 264.7, (b) HEK293 or (c) D556 cells based on iron concentrations measured by the 1,10 phenanthroline calorimetric assay. Cells were treated with IONR_(L), IONR_(S), or IONP at a Fe concentration of 50 μg Fe per mL for 4 h. Data are presented as mean values (n = 3) with standard deviations. Statistical significance of comparison (one-way ANOVA, Tukey's HSD test) is indicated as: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Non-significance is indicated as “ns”.

Fig. 3. Comparison of the cytotoxicity of oligosaccharide coated IONR_(L), IONR_(S), and spherical IONP on different cell lines measured by the MTT assay. (a) RAW264.7 (murine macrophage cells), (b) HEK293 (normal embryonic kidney cells) (c) D556 (medulloblastoma cells), and (d) MDA-MB-453 (triple negative breast cancer cells) were treated with IONR_(L), IONR_(S), and spherical IONP at different concentrations for 24 h. Data are presented as mean values (n = 3) with the standard deviations.

Fig. 4. Comparison of internalization and intracellular distributions of oligosaccharide coated IONP_(L), IONR_(S), and spherical IONP in different cell lines. Confocal fluorescence images of (a) RAW264.7, (b) HEK293, and (c) D556 were taken from cells treated with Cy7-labeled IONR_(L), IONR_(S), and IONP at a Fe concentration of 50 μg mL⁻¹ for 3 h. Cy7-labeled IONR and IONP – yellow, endosome/lysosome stained with lysotracker – red, and nucleus stained with Hoechst – blue. The cells without nanoparticle treatment were used as the control group. (d) Mean fluorescence intensity of cellular uptake and internalization of IONP_(L), IONR_(S), and IONP measured by ImageJ software and (e) percentages of co-localized signals from LysoTracker and IONP_(L), or IONR_(S), or IONP. The scale bar indicates 1 μm and 40× objective lens. Data are presented as mean values (n = 3) with the standard deviations. Statistical significance is noted as: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, when compared with values for the control and other inhibitors (one-way ANOVA, Tukey's HSD test). The values which are not significant are represented as “ns”.

Fig. 5. TEM images of selected cells with different oligosaccharide coated IONP and IONR internalized. (a–c) RAW246.7 cells with IONR_(L), IONR_(S), and IONP; (d–f) HEK293 cells with IONR_(L), IONR_(S), and IONP; (g–i) D556 cells with IONR_(L), IONR_(S), and IONP. The cells were incubated for 4 h with different nanoparticles at a 50 μg mL⁻¹ Fe concentration. Scale bar: 0.2 μm.

Fig. 6. Effects of endocytosis inhibitors on the cellular uptake of oligosaccharide coated IONR_(L), IONR_(S), and IONP in different cells. The cells were exposed to endocytosis inhibitors for 1 h in a serum-free medium, and then, incubated with fresh medium containing inhibitors and respective nanoparticles for 4 h. Inhibitors of clathrin (CPM) or caveolin (genistein) or lipid rafts (MβCD) macropinocytosis (amiloride) or dynamin (dynasore) or macropinocytosis/phagocytosis (CytoD) were used in the experiment. The intracellular Fe of nanoparticle treated (a, d, and g) RAW 264.7, (b, e, and h) HEK293 and (c, f, and i) D556 cells were measured. Data are presented as mean values (n = 3) with standard deviations. Statistical significance is noted as: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 when compared with values for the control and other inhibitors (one-way ANOVA, Tukey's HSD test).

Fig. 7. Selected TEM images of different oligosaccharide coated IONR and IONP compartmentalized in different cells at the 2 h time point after incubation and a nanoparticle concentration of 50 Fe μg mL⁻¹. Top row: RAW264.7 cells treated with oligosaccharide coated (a) IONR_(L), (b) IONR_(S), and (c) spherical IONP; center row: HEK293 cells treated with oligosaccharide coated (d) IONR_(L) (e) IONR_(S), and (f) spherical IONP; and bottom row: D556 cells treated with oligosaccharide coated (g) IONR_(L), (h) IONR_(S), and (i) IONP. Scale bar indicates 50 nm, 100 nm, 0.1 μm, and 0.2 μm. Green arrow – clathrin-mediated (clathrin-coated pits); brown arrow – caveolae-mediated (flask-shaped structures); blue arrow – macropinocytosis (macropinosomes); magenta arrow – phagocytosis.