Inert Coats of Magnetic Nanoparticles Prevent Formation of Occlusive Intravascular Co-aggregates With Neutrophil Extracellular Traps

Abstract

If foreign particles enter the human body, the immune system offers several mechanisms of response. Neutrophils forming the first line of the immune defense either remove pathogens by phagocytosis, inactivate them by degranulation or release of reactive oxygen species or immobilize them by the release of chromatin decorated with the granular proteins from cytoplasm as neutrophil extracellular traps (NETs). Besides viable microbes like fungi, bacteria or viruses, also several sterile inorganic particles including nanoparticles reportedly activate NET formation. The physicochemical nanoparticle characteristics fostering NET formation are still elusive. Here we show that agglomerations of non-stabilized superparamagnetic iron oxide nanoparticles (SPIONs) induce NET formation by isolated human neutrophils, in whole blood experiments under static and dynamic conditions as well as in vivo. Stabilization of nanoparticles with biocompatible layers of either human serum albumin or dextran reduced agglomeration and NET formation by neutrophils. Importantly, this passivation of the SPIONs prevented vascular occlusions in vivo even when magnetically accumulated. We conclude that higher order structures formed during nanoparticle agglomeration primarily trigger NET formation and the formation of SPION-aggregated NET-co-aggregates, whereas colloid-disperse nanoparticles behave inert and are alternatively cleared by phagocytosis.

Methods of Granulocyte Isolation from Human Blood and Labeling with Multimodal Superparamagnetic Iron Oxide Nanoparticles

“…However, other studies [53,58,60] reported SPION incubation times up to 24 h. Previous studies have reported that SPION concentrations between 1 and 200 µg Fe/mL showed cellular viability greater than 86% [51,[57][58][59][60]85,87,88], whereas cellular viability was reduced at concentrations from 5 to 20 mg Fe/mL [53,54,86]. The dextran coating used in M-SPIONs has been reported as adequate for biomedical applications in terms of safety and biocompatibility [89] and is used in most granulocyte-labeling studies to cover nanoparticles [51,57,[59][60][61]88].…”

“…For internalization, it is best for the nanoparticles to have a positive surface charge (zeta potential), given the negative surface charge of granulocytes [75,82] and the electrostatic process involved [77,83,84]. Therefore, granulocyte and leukocyte-labeling studies that have used SPIONs with a negative zeta potential (0 to −53 mV) [51,54,57,58,85,86] have reported the necessity of using chemical or physical transfection agents or other strategies that would contribute to SPION internalization into cells and boost the internalization yield [51,54,57,86].…”

“…In our study, we found satisfactory viability values using a 4-h labeling time (99.3%, 99.6% and 98.6% for the respective M-SPION concentrations of 10, 30 and 50 µg Fe/mL). In studies on granulocyte and leukocyte-labeling with SPIONs, the incubation time was reported to be between 1 and 4 h for labeling [51,54,57,59,61,62,85,86]. For example, in the study of Tang et al [61], an incubation time of 3 h was best for the clinical application of these cells.…”

“…In most studies, SPION internalization into granulocytes or leukocytes is confirmed using Prussian blue staining and MRI [51,54,57,61], because the composition of nanoparticles is based on iron oxide. However, as the nanoparticles used in our study have multimodal features (magnetic/dual fluorescence), it was possible to verify internalization using Prussian blue staining and MRI, as well as using fluorescence images in the visible spectrum (rhodamine) and infrared spectrum (IR750).…”

“…Most studies employing exogenously labeled granulocytes to analyze infection or inflammation processes have used SPIONs with different strategies [51,54,[57][58][59][60][61][62]. SPIONs, when coupled with near-infrared spectrum fluorophores, enhance pathological assessments due to the sensitivity or specificity of the complementary information provided by the optical imaging modality [63], enabling multimodal imaging [64,65].…”