Associate Professor University of Tennessee, Knoxville Knoxville, Tennessee, United States
Introduction: Poly-ethylene-glycol (PEG) and poly-ethylene-oxide (PEO) nanoparticles (NPs) - including cylindrical micelles (CNPs), spherical micelles (SNPs), and PEGylated liposomes (PLs) - are hypothesized to be cleared in vivo by opsonization followed by liver macrophage phagocytosis. This hypothesis has been used to explain the rapid and significant localization of NPs to the liver after administration into the mammalian vasculature. But the rapid localization of these NPs to the liver after injection into the vasculature occurs before vertebrates can make antibodies against the chemical moieties of the NP. The time scale would be days for IgM production and a week for IgG production. Thus, additional mechanisms must be responsible for NP localization to the liver. The two postulated mechanisms are protein corona formation on the NP and/or direct binding of the NP to macrophage surface receptors. Here, we test this postulate.
Materials and
Methods: We used a combination of in vitro and in vivo experiments to determine how PEG/PEO-NPs bound macrophages and how the PEG/PEO-NPs affected macrophage transcription. We used classic binding experiments and size exclusion chromatography to determine which serum proteins bound PEG/PEO-NPs. We used scid-gamma mice which cannot make antibodies to determine the effects of antibodies on PEG/PEO-NP biodistribution. We controlled the protein content of the media in which the macrophages where cultured to show how proteins in serum affect the binding of PEG/PEO-NPs to macrophages. We used flow cytometry to quantify the binding of PEG/PEO-NPs to macrophages. We used bulk RNA sequencing to determine the effects of PEG/PEO-NPs on macrophage transcription.
Results, Conclusions, and Discussions: We show that the opsonization-phagocytosis nexus is not the major factor driving PEG/PEO-NP - macrophage interactions. First, mouse and human blood proteins had insignificant affinity for PEG/PEO-NPs. Second, PEG/PEO-NPs bound macrophages in the absence of serum proteins. Third, lipoproteins blocked PEG/PEO-NP binding to macrophages. Because of these findings, we tested the postulate that PEG/PEO-NPs bind (apo)lipoprotein receptors. Indeed, PEG/PEO-NPs triggered an in vitro macrophage transcription program that was similar to that triggered by lipoproteins and different from that triggered by lipopolysaccharide (LPS) and group A Streptococcus. Unlike LPS and pathogens, PLs did not increase transcripts involved in phagocytosis or inflammation. High-density lipoprotein (HDL) and SNPs triggered remarkably similar mouse bone-marrow-derived macrophage transcription programs. Unlike opsonized pathogens, CNPs, SNPs, and PLs lowered macrophage autophagosome levels and either reduced or did not increase the secretion of key macrophage pro-inflammatory cytokines and chemokines. Thus, the sequential opsonization and phagocytosis process is likely a minor aspect of PEG/PEO-NP - macrophage interactions that occur rapidly after injection into the vasculature of mammals. Instead, PEG/PEO-NP interactions with (apo)lipoprotein and scavenger receptors appear to be a strong driving force for PEG/PEO-NP - macrophage binding, entry, and downstream effects. We hypothesize that the high presence of scavenger receptors on liver macrophages and on liver sinusoidal endothelial cells is the reason PEG/PEO-NPs localize rapidly and strongly to the liver.
