Rapid cellular uptake of citrate-coated iron oxide nanoparticles unaffected by cell-surface glycosaminoglycans.

Citrate-coated iron oxide nanoparticles, specifically Synomag®-COOH (SynC), are promising tracers in magnetic particle imaging (MPI) due to their high magnetic moments and rapid cellular uptake. The mechanisms driving efficient SynC uptake remain unclear. Previous observations suggest a role of the extracellular glycocalyx during nanoparticle uptake. Here, we ascertain whether the cell-surface glycosaminoglycans (GAGs) regulate the uptake of SynC. Using transmission electron microscopy (TEM), we visualized SynC uptake by THP-1 cells, a human acute monocytic leukemia cell line. We investigated the interaction of SynC with GAGs in living cells using click-chemistry-based labeling. Upon treating THP-1 cells with chondroitinase or hyaluronidase and with a xylosyltransferase-deficient cell line, we quantified SynC uptake and measured interactions of SynC with cells in real time using magnetic particle spectroscopy (MPS). The THP-1 cell membrane engulfed or formed extensions around SynC, indicating uptake through pinocytosis and phagocytosis. We measured an increased MPS signal of SynC within seconds of cell contact, suggesting an interaction with extracellular components like the glycocalyx. Upon adding SynC to THP-1 cells, we could not observe disruption of fluorescently labeled GAGs or an enhanced intracellular fluorescence, implying that SynC does not accelerate the turnover of GAGs by binding. Lack of chondroitin sulfate, heparan sulfate, and hyaluronic acid did not affect the rapid magnetic behavior increase of SynC upon cell contact. Accordingly, we measured no significant differences in SynC uptake between wild type cells and our GAG-deficient models. These findings suggest that GAGs act as a permeable bandpass for SynC nanoparticles with a minor negative surface charge of -13.8 mV. This finding has significant implications for MPI-based cell tracking because it facilitates efficient tracking of cell types that lack a strong repulsion by cell-surface GAGs. It will be crucial to investigate whether the rapid uptake of SynC is cell-type specific and influenced by different extracellular matrix compositions.

Lack of Laminar Shear Stress Facilitates the Endothelial Uptake of Very Small Superparamagnetic Iron Oxide Nanoparticles by Modulating the Endothelial Surface Layer.

Purpose: To study whether the absence of laminar shear stress (LSS) enables the uptake of very small superparamagnetic iron oxide nanoparticles (VSOP) in endothelial cells by altering the composition, size, and barrier function of the endothelial surface layer (ESL). Methods and Results: A quantitative particle exclusion assay with living human umbilical endothelial cells using spinning disc confocal microscopy revealed that the dimension of the ESL was reduced in cells cultivated in the absence of LSS. By combining gene expression analysis, flow cytometry, high pressure freezing/freeze substitution immuno-transmission electron microscopy, and confocal laser scanning microscopy, we investigated changes in ESL composition. We found that increased expression of the hyaluronan receptor CD44 by absence of shear stress did not affect the uptake rate of VSOPs. We identified collagen as a previously neglected component of ESL that contributes to its barrier function. Experiments with inhibitor halofuginone and small interfering RNA (siRNA) demonstrated that suppression of collagen expression facilitates VSOP uptake in endothelial cells grown under LSS. Conclusion: The absence of laminar shear stress disturbs the barrier function of the ESL, facilitating membrane accessibility and endocytic uptake of VSOP. Collagen, a previously neglected component of ESL, contributes to its barrier function.

Immuno-Electron and Confocal Laser Scanning Microscopy of the Glycocalyx.

The glycocalyx (GCX), a pericellular carbohydrate rich hydrogel, forms a selective barrier that shields the cellular membrane, provides mechanical support, and regulates the transport and diffusion of molecules. The GCX is a fragile structure, making it difficult to study by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). Sample preparation by conventional chemical fixation destroys the GCX, giving a false impression of its organization. An additional challenge is to process the GCX in a way that preserves its morphology and enhanced antigenicity to study its cell-specific composition. The aim of this study was to provide a protocol to preserve both antigen accessibility and the unique morphology of the GCX. We established a combined high pressure freezing (HPF), osmium-free freeze substitution (FS), rehydration, and pre-embedding immunogold labeling method for TEM. Our results showed specific immunogold labeling of GCX components expressed in human monocytic THP-1 cells, hyaluronic acid receptor (CD44) and chondroitin sulfate (CS), and maintained a well-preserved GCX morphology. We adapted the protocol for antigen localization by CLSM and confirmed the specific distribution pattern of GCX components. The presented combination of HPF, FS, rehydration, and immunolabeling for both TEM and CLSM offers the possibility for analyzing the morphology and composition of the unique GCX structure.