The Long-Term Impact of Polysaccharide-Coated Iron Oxide Nanoparticles on Inflammatory-Stressed Mice

Abstract

Since iron oxide nanoparticles (IONPs) are expected to be important tools in medical care, patients with inflammatory diseases will be increasingly exposed to IONPs in the future. Here, we assessed the short- and long-term impact of polysaccharide (PS)-coated IONPs on mice with persistent systemic inflammation. To this end, PS-IONPs were synthetized by a core-shell method. Mice were regularly injected with sterile zymosan. PS-IONPs were administered intravenously. At specific nanoparticle injection post-observation times, the organ iron concentration was determined via atomic absorption spectrometry, the expression of NF-κB-related proteins using SDS-PAGE and immunoblotting, as well as body weight and haemograms. Finally, the mediator secretion in blood plasma was analysed using multiplexed ELISA. Our data show that PS-IONPs induce short-term changes of iron levels in distinct organs and of NF-κB p65 and p50, p100, COX-2s, and Bcl-2 protein expression in the liver of inflammatory stressed mice. In the long term, there was an attenuated expression of several NF-κB-related proteins and attenuated features of inflammatory-based anaemia in blood. PS-IONPs weakly influenced the blood cytokine levels. PS-IONPs are biocompatible, but given their short-term pro-inflammatory impact, they should prospectively be applied with caution in patients with inflammatory diseases of the liver.

Keywords: NF-kappaB; health; iron oxide nanoparticles; long-term biocompatibility; long-term toxicity; metabolism; nanomedicine; polysaccharides; theranostic.

Figure 1

The time-dependent interventions in mice and principal endpoints during in vivo experimentation. Frequent subcutaneous injections of zymosan into the right hind leg (7 cycles of 18 mg/kg body weight each) were carried out to induce a chronic inflammatory state. PS-IONPs: intravenous injection of 50 µmol Fe/kg body weight.

Figure 2

Level of edema as a result of subcutaneous injection of zymosan into mice of the “+/+” animal group. As a component of the cell wall from Saccharomyces cerevisiae, zymosan is known to activate various immune cells, which release cellular mediators promoting vascular permeability. Arrow: exemplary anatomical location of local edema at their right hind legs. The animal pictures refer to different time-points (in months, highlighted as coloured boxes) and are representative for all animals of the group. For clarity reasons, we do not depict the standard deviations of the mean in the graph.

Figure 3

Short-term effects of PS-IONPs on the iron distribution in the organs of mice with a persistent inflammatory state in comparison to the control groups (0.25 months after intravenous application). Experimental group: “+/+”—animals with a persistent inflammatory state (zymosan: 7 cycles of 18 µg/kg body weight, PS-IONPs: 50 µmol Fe/kg body weight). Control groups: “−/−”—animals without persistent inflammation and no intravenous PS-IONP injection, and “−/+”—animals without persistent inflammation but with intravenous injection of PS-IONPs. Data are represented as iron content in milligrams per gram of dry tissue mass (individual data points), along with the mean and standard deviation; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (t-test with Welch’s correction).

Figure 4

Long-term impact of PS-IONPs on the iron distribution in the organs of mice with persistent inflammatory and intravenously injected PS-IONPs in comparison to the control groups. Experimental group: “+/+”—animals with a persistent inflammatory state (zymosan: 7 cycles of 18 µg/kg body weight, PS-IONPs: 50 µmol Fe/kg body weight). Control groups: “−/−”—animals without persistent inflammation and no intravenous PS-IONP injection, and “−/+”—animals without persistent inflammation but with intravenous injection of PS-IONPs, “+/−”—animals with persistent inflammation but without intravenous injection of PS-IONPs. Data are represented as iron content in milligrams per gram of dry tissue mass (individual data points). Only regression lines with R2 larger than 0.12 and with slopes significantly nonzero with p < 0.05 were depicted (red lines). # not determined.

Figure 5

Long-term impact of expression of PS-IONPs on NF-κB nuclear factor protein expression in the liver of mice with a persistent inflammatory state in comparison to the control groups. Experimental group: “+/+”—animals with a persistent inflammatory state (zymosan: 7 cycles of 18 µg/kg body weight, PS-IONPs: 50 µmol Fe/kg body weight). Control groups: “−/−”—animals without persistent inflammation and no intravenous PS-IONP injection, and “−/+”—animals without persistent inflammation but with intravenous injection of PS-IONPs, “+/−”—animals with persistent inflammation but without intravenous injection of PS-IONPs. Protein expression is presented relative to the housekeeping proteins β-actin or GAPDH (dots) and as mean and standard deviation of the mean; * p < 0.05, ** p < 0.01 (Mann–Whitney U-test), # not determined.

Figure 6

Long-term impact of PS-IONPs on NF-κB regulator protein expression in the liver of mice with a persistent inflammatory state in comparison to the control groups Experimental group: “+/+”—animals with a persistent inflammatory state (zymosan: 7 cycles of 18 µg/kg body weight, PS-IONPs: 50 µmol Fe/kg body weight). Control groups: “−/−”—animals without persistent inflammation and no intravenous PS-IONP injection, and “−/+”—animals without persistent inflammation but with intravenous injection of PS-IONPs, “+/−”—animals with persistent inflammation but without intravenous injection of PS-IONPs. Protein expression is presented relative to the housekeeping proteins β-actin or GAPDH (dots) and as mean and standard deviation of the mean; * p < 0.05, ** p < 0.01 (Mann–Whitney U-test), # not determined.

Figure 7

Long-term impact of PS-IONPs on NF-κB downstream effector protein expression in the liver of mice with a persistent inflammatory state in comparison to the control groups. Experimental group: “+/+”—animals with a persistent inflammatory state (zymosan: 7 cycles of 18 µg/kg body weight, PS-IONPs: 50 µmol Fe/kg body weight). Control groups: “−/−”—animals without persistent inflammation and no intravenous PS-IONP injection, and “−/+”—animals without persistent inflammation but with intravenous injection of PS-IONPs, “+/−”—animals with persistent inflammation but without intravenous injection of PS-IONPs. Protein expression is presented relative to the housekeeping proteins β-actin or GAPDH (dots) and as mean and standard deviation of the mean; * p < 0.05, ** p < 0.01 (Mann–Whitney U-test), # not determined.

Figure 8

Cytokine levels of blood plasma with pro-inflammatory potential. Experimental group: “+/+”—animals with a persistent inflammatory state (zymosan: 7 cycles of 18 µg/kg body weight, PS-IONPs: 50 µmol Fe/kg body weight). Control groups: “−/−”—animals without persistent inflammation and no intravenous PS-IONP injection, and “−/+”—animals without persistent inflammation but with intravenous injection of PS-IONPs, “+/−”—animals with persistent inflammation but without intravenous injection of PS-IONPs. Data are plotted as mean and standard deviation of the mean of 3 to 5 animals per group, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way ANOVA with Tukey’s multiple comparisons test).

Figure 9

Simplified organ map showing the systemic influence of PS-IONPs in animals with persistent inflammation. (A) Immediately after intravenous injection of PS-IONPs, (B) short-term situation (2 to 4 months thereafter), and (C) long-term situation (4 to 6 months thereafter). pi: pro-inflammatory impact of PS-IONPs in the liver after extensive accumulation in Kupffer cells, ai: anti-inflammatory impact of PS-IONPs in the liver, MPS: organs of the mononuclear phagocyte system. Created with BioRender.com.