Intravenous Injection of PEI-Decorated Iron Oxide Nanoparticles Impacts NF-kappaB Protein Expression in Immunologically Stressed Mice

Abstract

Nanoparticle-based formulations are considered valuable tools for diagnostic and treatment purposes. The surface decoration of nanoparticles with polyethyleneimine (PEI) is often used to enhance their targeting and functional properties. Here, we aimed at addressing the long-term fate in vivo and the potential "off-target" effects of PEI decorated iron oxide nanoparticles (PEI-MNPs) in individuals with low-grade and persistent systemic inflammation. For this purpose, we synthesized PEI-MNPs (core-shell method, PEI coating under high pressure homogenization). Further on, we induced a low-grade and persistent inflammation in mice through regular subcutaneous injection of pathogen-associated molecular patterns (PAMPs, from zymosan). PEI-MNPs were injected intravenously. Up to 7 weeks thereafter, the blood parameters were determined via automated fluorescence flow cytometry, animals were euthanized, and the organs analyzed for iron contents (atomic absorption spectrometry) and for expression of NF-κB associated proteins (p65, IκBα, p105/50, p100/52, COX-2, Bcl-2, SDS-PAGE and Western blotting). We observed that the PEI-MNPs had a diameter of 136 nm and a zeta-potential 56.9 mV. After injection in mice, the blood parameters were modified and the iron levels were increased in different organs. Moreover, the liver of animals showed an increased protein expression of canonical NF-κB signaling pathway members early after PEI-MNP application, whereas at the later post-observation time, members of the non-canonical signaling pathway were prominent. We conclude that the synergistic effect between PEI-MNPs and the low-grade and persistent inflammatory state is mainly due to the hepatocytes sensing infection (PAMPs), to immune responses resulting from the intracellular metabolism of the uptaken PEI-MNPs, or to hepatocyte and immune cell communications. Therefore, we suggest a careful assessment of the safety and toxicity of PEI-MNP-based carriers for gene therapy, chemotherapy, and other medical applications not only in healthy individuals but also in those suffering from chronic inflammation.

Keywords: NF-kappaB; inflammation; iron oxide nanoparticles; polyethyleneimine.

Figure 1

Experimental setup showing the time-dependent interventions into animals during experimentation. Repeated subcutaneous injection of zymosan into the right hind leg (3 cycles of 18 mg/kg body weight each) to induce a low-grade and persistent inflammatory state. MNP: magnetic nanoparticles decorated with polyethyleneimine (PEI), intravenous injection of 50 µmol Fe/kg body weight, 700 µg PEI per mg iron.

Figure 2

Iron concentrations in organs of animals with low-grade persistent inflammatory state at one (1) week after intravenous application of PEI-MNPs. Experimental groups: “+/+”: animals with low-grade persistent inflammation state (subcutaneous injection of zymosan, 3 times 18 µg/kg body weight) and with intravenous injection of PEI-MNPs (50 µmol Fe/kg body weight, 700 µg PEI per mg iron); “−/−”: animals without low-grade persistent inflammation state and without intravenous injection of PEI-MNPs; “−/+”: animals without low-grade persistent inflammation state but with intravenous injection with PEI-MNP. Data is plotted as mg iron per g dry tissue mass, and as mean and standard deviation of the mean, n = 3 to 5 animals per group, * p < 0.05, ** p < 0.01 (t-test with Welch’s correction).

Figure 3

Iron concentrations in the organs of animals with low-grade persistent inflammatory state at seven (7) weeks after intravenous application of PEI-MNPs. Experimental groups: “+/+”: animals with low-grade persistent inflammation state (subcutaneous injection of zymosan, 3 times 18 µg/kg body weight) and with intravenous injection of PEI-MNPs (50 µmol Fe/kg body weight, 700 µg PEI per mg iron); “−/−”: animals without low-grade persistent inflammation state and without intravenous injection of PEI-MNPs, “−/+”: animals without low-grade persistent inflammation state but with intravenous injection of PEI-MNP, “+/−”: animals with low-grade persistent inflammation state but without intravenous injection of PEI-MNP. Data are plotted as mg iron per g dry tissue mass, and as mean and standard deviation of the mean, n = 3 to 5 animals per group, * p < 0.05 (t-test with Welch’s correction).

Figure 4

Protein expression of the important players of the NF-κB signaling pathway in the liver of animals with low-grade persistent inflammatory state after one (1) week after intravenous application of PEI-MNPs. Experimental groups: “+/+”: animals with low-grade persistent inflammation state (subcutaneous injection of zymosan, 3 times 18 µg/kg body weight) and with intravenous injection of PEI-MNPs (50 µmol Fe/kg body weight, 700 µg PEI per mg iron); “−/−”: animals without low-grade persistent inflammation state and without intravenous injection of PEI-MNPs; “−/+”: animals without low-grade persistent inflammation state but with intravenous injection of PEI-MNP; “+/−”: animals with low-grade persistent inflammation state but without intravenous injection of PEI-MNP. Protein expression normalized to the housekeeping cellular protein β-actin. NF-κB nuclear factors in red, regulators in green and effector proteins in blue. Data are plotted as mean and standard deviation of the mean, n = 5 animals per group, * p < 0.05, ** p < 0.01 (Mann–Whitney U test).

Figure 5

Increased protein expression of important players of the NF-κB pathway in the liver of animals with low-grade persistent inflammatory state at 7 weeks after intravenously injected PEI-MNP. Experimental groups: “+/+”: animals with low-grade persistent inflammation state (subcutaneous injection of zymosan, 3 times 18 µg/kg body weight) and with intravenous injection of PEI-MNPs (50 µmol Fe/kg body weight, 700 µg PEI per mg iron); “−/−”: animals without low-grade persistent inflammation state and without intravenous injection of PEI-MNPs; “−/+”: animals without low-grade persistent inflammation state but with intravenous injection of PEI-MNP. Protein expression normalized to the housekeeping cellular protein β-actin. NF-κB nuclear factors in red, regulators in green and effector proteins in blue. Data are plotted as mean and standard deviation of the mean, n = 5 animals per group. * p < 0.05, ** p < 0.01 (Mann–Whitney U test).

Figure 6

Hemogram of animals with low-grade persistent inflammatory state in comparison with controls after intravenous application of PEI-MNPs. Experimental groups: “+/+”: animals with low-grade persistent inflammation state (subcutaneous injection of zymosan, 3 times 18 µg/kg body weight) and with intravenous injection of PEI-MNPs (50 µmol Fe/kg body weight, 700 µg PEI per mg iron; “−/−”: animals without low-grade persistent inflammation state and without intravenous injection of PEI-MNPs; “−/+”: animals without low-grade persistent inflammation state but with intravenous injection of PEI-MNP. WBC: white blood cells, RBC: red blood cells, HGB: hemoglobin, HCT: hematocrit MCV: red blood cell mean volume, MCH: mean red blood cell hemoglobin, MCHC: mean cell hemoglobin concentration. Data are plotted as mean and standard deviation of the mean, n = 5 animals per group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (unpaired t-test with Welch’s correction).

Figure 7

Proposed mechanism of the mutual effect of PEI-MNPs on both the tissue-resident macrophage hepatocytes during a systemic low-grade and persistent inflammatory state. Hepatocytes sense the pro-inflammatory cytokines from tissue-resident macrophages after uptake of PEI-MNPs.