Differential Nanoparticle Sequestration by Macrophages and Scavenger Endothelial Cells Visualized in Vivo in Real-Time and at Ultrastructural Resolution

Abstract

Despite the common knowledge that the reticuloendothelial system is largely responsible for blood clearance of systemically administered nanoparticles, the sequestration mechanism remains a “black box”. Using transgenic zebrafish embryos with cell type-specific fluorescent reporters and fluorescently labeled model nanoparticles (70 nm SiO2), we here demonstrate simultaneous three-color in vivo imaging of intravenously injected nanoparticles, macrophages, and scavenger endothelial cells (SECs). The trafficking processes were further revealed at ultrastructural resolution by transmission electron microscopy. We also find, using a correlative light-electron microscopy approach, that macrophages rapidly sequester nanoparticles via membrane adhesion and endocytosis (including macropinocytosis) within minutes after injection. In contrast, SECs trap single nanoparticles via scavenger receptor-mediated endocytosis, resulting in gradual sequestration with a time scale of hours. Inhibition of the scavenger receptors prevented SECs from accumulating nanoparticles but enhanced uptake in macrophages, indicating the competitive nature of nanoparticle clearance in vivo. To directly quantify the relative contributions of the two cell types to overall nanoparticle sequestration, the differential sequestration kinetics was studied within the first 30 min post-injection. This revealed a much higher and increasing relative contribution of SECs, as they by far outnumber macrophages in zebrafish embryos, suggesting the importance of the macrophage:SECs ratio in a given tissue. Further characterizing macrophages on their efficiency in nanoparticle clearance, we show that inflammatory stimuli diminish the uptake of nanoparticles per cell. Our study demonstrates the strength of transgenic zebrafish embryos for intravital real-time and ultrastructural imaging of nanomaterials that may provide mechanistic insights into nanoparticle clearance in rodent models and humans.

Tracing the In Vivo Fate of Nanoparticles with a “Non-Self” Biological Identity

“…As previously 25 , nanoparticles with pre-formed FBS PC were IV injected at the common cardinal vein that leads to similar biodistribution as that of Pristine but with extended sequestration in veins ( Fig. 2a and Supplementary Fig.…”

“…Next, we have chosen 15 min as the minimal duration for the incubation of FBS PC nanoparticles in DrBP to characterize PC retention in vitro, corresponding to previously estimated elimination half-life of 14 min following IV injection of the same SiO 2 nanoparticles but without pre-formed FBS PC 25 . Fluorescence labelling and MS/MS approaches confirmed retention of APOA1 in the FBS PC while Fetuin-A was added from the 15 min incubation in DrBP (Fig.…”

“…Zebrafish embryos have recently emerged as a bioimaging model for preclinical screening of nanomedicines [15][16][17][18][19][20][21][22][23] with a unique opportunity of whole-embryo fixation for transmission electron microscopy (TEM) to visualize intracellular trafficking of nanoparticles in vivo at high resolution 24,25 . Here we used the zebrafish model to unravel the in vivo journey of 70 nm SiO 2 nanoparticles with pre-formed FBS PC and the pathophysiological consequences.…”

“…The nanoparticles represent a model system especially suited for this study owing to the matrix-incorporated fluorescence tracers of choice, intrinsic high electron density of SiO 2 , and the large particle size that allows excellent recovery of the nanoparticle-protein complexes even after extensive washing by centrifugation. We have previously established an intravital real-time imaging approach for the same SiO 2 nanoparticles but without pre-formed FBS PC (hereafter referred to as "Pristine nanoparticles") 25 . Of note is the scavenger phenotype of venous endothelial cells (ECs) lining the caudal vein (CV) and the CV plexus, the functional analogue of liver sinusoidal ECs in mammals 16 , which continuously sequestered blood-borne nanoparticles in a scavenger receptor-dependent manner 25 with blood clearance kinetics resembling that of mammalian models 26 .…”

“…We have previously established an intravital real-time imaging approach for the same SiO 2 nanoparticles but without pre-formed FBS PC (hereafter referred to as "Pristine nanoparticles") 25 . Of note is the scavenger phenotype of venous endothelial cells (ECs) lining the caudal vein (CV) and the CV plexus, the functional analogue of liver sinusoidal ECs in mammals 16 , which continuously sequestered blood-borne nanoparticles in a scavenger receptor-dependent manner 25 with blood clearance kinetics resembling that of mammalian models 26 . Macrophages, on the other hand, engaged blood-borne nanoparticles rapidly via membrane adhesion and constitutive macropinocytosis independently of scavenger receptors and without pro-inflammatory responses 25 .…”